Compositions and methods for drug delivery using pH sensitive molecules

ABSTRACT

A system relating to the delivery of desired compounds (e.g., drugs and nucleic acids) into cells using pH-sensitive delivery systems. The system provides compositions and methods for the delivery and release of a compound to a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of prior provisional applications60/137,859 filed on Jun. 7, 1999, 60/167,836 filed on Nov. 29, 1999 and60/172,809 filed on Dec. 21, 1999 and is a divisional application ofSer. No. 09/589,978 filed on Jun. 7, 2000 now U.S. Pat. No. 6,630,351.

FIELD OF THE INVENTION

The present invention relates to the delivery of desired compounds(e.g., drugs and nucleic acids) into cells using pH-sensitive deliverysystems. The present invention provides compositions and methods for thedelivery and release of a compound of interest to a cell.

BACKGROUND OF THE INVENTION

Drug Delivery

A variety of methods and routes of administration have been developed todeliver pharmaceuticals that include small molecular drugs andbiologically active compounds such as peptides, hormones, proteins, andenzymes to their site of action. Parenteral routes of administrationinclude intravascular (intravenous, intraarterial), intramuscular,intraparenchymal, intradermal, subdermal, subcutaneous, intratumor,intraperitoneal, and intralymphatic injections that use a syringe and aneedle or catheter. The blood circulatory system provides systemicspread of the pharmaceutical. Polyethylene glycol and other hydrophilicpolymers have provided protection of the pharmaceutical in the bloodstream by preventing its interaction with blood components and toincrease the circulatory time of the pharmaceutical by preventingopsonization, phagocytosis and uptake by the reticuloendothelial system.For example, the enzyme adenosine deaminase has been covalently modifiedwith polyethylene glycol to increase the circulatory time andpersistence of this enzyme in the treatment of patients with adenosinedeaminase deficiency.

The controlled release of pharmaceuticals after their administration isunder intensive development. Pharmaceuticals have also been complexedwith a variety of biologically-labile polymers to delay their releasefrom depots. These polymers have included copolymers ofpoly(lactic/glycolic acid) (PLGA) (Jain, R. et al. Drug Dev. Ind. Pharm.24, 703–727 (1998), ethylvinyl acetate/polyvinyl alcohol (Metrikin, D Cand Anand, R, Curr Opin Ophthalmol 5, 21–29, 1994) as typical examplesof biodegradable and non-degradable sustained release systemsrespectively.

Transdermal routes of administration have been effected by patches andionotophoresis. Other epithelial routes include oral, nasal,respiratory, and vaginal routes of administration. These routes haveattracted particular interest for the delivery of peptides, proteins,hormones, and cytokines, which are typically administered by parenteralroutes using needles. For example, the delivery of insulin viarespiratory, oral, or nasal routes would be very attractive for patientswith diabetes mellitus. For oral routes, the acidity of the stomach (pHless than 2) is avoided for pH-sensitive compounds by concealingpeptidase-sensitive polypaptides inside pH-sensitive hydrogel matrix(copolymers of polyethyleneglycol and polyacrylic acid). After passinglow pH compartments of gastrointestinal tract such structures swells athigher pH releasing thus a bioactive compound (Lowman A M et al. J.Pharm. Sci. 88, 933–937 (1999). Capsules have also been developed thatrelease their contents within the small intestine based uponpH-dependent solubility of a polymer. Copolymers of polymethacrylic acid(Eudragit S, Rohm America) are known as polymers which are insoluble atlower pH but readily solubilized at higher pH, so they are used asenteric coatings (Z Hu et al. J. Drug Target., 7, 223, 1999).

Biologically active molecules may be assisted by a reversible formationof covalent bonds. Quite often, it is found that the drug administeredto a patient is not the active form of the drug, but is what is a calleda prodrug that changes into the actual biologically active compound uponinteractions with specific enzymes inside the body. In particular,anticancer drugs are quite toxic and are administered as prodrugs whichdo not become active until they come in contact with the cancerous cell(Sezaki, I I., Takakura, Y., Hashida, M. Adv. Drug. Delivery Reviews 3,193, 1989).

Recent studies have found that pH in solid tumors is 0.5 to 1 unitslower than in normal tissue (Gerweck L E et al. Cancer Res. 56, 1194(1996). Hence, the use of pH-sensitive polymers for tumor targeting isjustified. However, this approach was demonstrated only in vitro(Berton, M, Eur. J. Pharm. Biopharm. 47, 119–23, 1999).

Liposomes were also used as drug delivery vehicles for low molecularweight drugs and macromolecules such as amphotericin B for systemicfungal infections and candidiasis. Inclusion of anti-cancer drugs suchas adriamycin have been developed to increase their delivery to tumorsand reduce it to other tissue sites (e.g. heart) thereby decreasingtheir toxicity. pH-sensitive polymers have been used in conjunction withliposomes for the triggered release of an encapsulated drug. Forexample, hydrophobically-modified N-isopropylacrylamide-methacrylic acidcopolymer can render regular egg phosphatidyl chloline liposomespH-sensitive by pH-dependent interaction of grafted aliphatic chainswith lipid bilayer (O Meyer et al., FEBS Lett., 421, 61, 1998).

Gene and Nucleic Acid-Based Delivery

Gene or polynucleotide transfer is the cardinal process of gene therapy.The gene needs to be transferred across the cell membrane and enter thenucleus where the gene can be expressed. Gene transfer methods currentlybeing explored included viral vectors and physical-chemical methods.

Viruses have evolved over millions of year to transfer their genes intomammalian cells. Viruses can be modified to carry a desired gene andbecome a “vector” for gene therapy. Using standard recombinanttechniques, the harmful or superfluous viral genes can be removed andreplaced with the desired normal gene. This was first accomplished withmouse retroviruses. The development of retroviral vectors were thecatalyst that promoted current gene therapy efforts. However, theycannot infect all cell types very efficiently, especially in vivo. Otherviral vectors based on Herpes virus are being developed to enable moreefficient gene transfer into brain cells. Adenoviral and adenoassociatedvectors are being developed to infect lung and other cells.

Besides using viral vectors, it is possible to directly transfer genesinto mammalian cells. Usually, the desired gene is placed withinbacterial plasmid DNA along with a mammalian promoter, enhancer, andother sequences that enable the gene to be expressed in mammalian cells.Several milligrams of the plasmid DNA containing all these sequences canbe prepared and purified from the bacterial cultures. The plasmid DNAcontaining the desired gene can be incorporated into lipid vesicles(liposomes including cationic lipids such as Lipofectin) that thentransfer the plasmid DNA into the target cell. Plasmid DNA can also becomplexed with proteins that target the plasmid DNA to specific tissuesjust as certain proteins are taken up (endocytosed) by specific cells.Also, plasmid DNA can be complexed with polymers such as polylysine andpolyethylenimine. Another plasmid-based technique involves “shooting”the plasmid DNA on small gold beads into the cell using a “gun”.Finally, muscle cells in vivo have the unusual ability to take up andexpress plasmid DNA.

Gene therapy approaches can be classified into direct and indirectmethods. Some of these gene transfer methods are most effective whendirectly injected into a tissue space. Direct methods using many of theabove gene transfer techniques are being used to target tumors, muscle,liver, lung, and brain. Other methods are most effective when applied tocells or tissues that have been removed from the body and thegenetically-modified cells are then transplanted back into the body.Indirect approaches in conjunction with retroviral vectors are beingdeveloped to transfer genes into bone marrow cells, lymphocytes,hepatocytes, myoblasts and skin cells.

Gene Therapy and Nucleic Acid-Based Therapies

Gene therapy promises to be a revolutionary advance in the treatment ofdisease. It is a fundamentally new approach for treating disease that isdifferent from the conventional surgical and pharmaceutical therapies.Conceptually, gene therapy is a relatively simple approach. If someonehas a defective gene, then gene therapy would fix the defective gene.The disease state would be modified by manipulating genes instead oftheir products, i.e. proteins, enzymes, enzyme substrates and enzymeproducts. Although, the initial motivation for gene therapy was thetreatment of genetic disorders, it is becoming increasingly apparentthat gene therapy will be useful for the treatment of a broad range ofacquired diseases such as cancer, infectious disorders (AIDS), heartdisease, arthritis, and neurodegenerative disorders (Parkinson's andAlzheimer's).

Gene therapy promises to take full-advantage of the major advancesbrought about by molecular biology. While, biochemistry is mainlyconcerned with how the cell obtains the energy and matter that isrequired for normal function, molecular biology is mainly concerned withhow the cell gets the information to perform its functions. Molecularbiology wants to discover the flow of information in the cell. Using themetaphor of computers, the cell is the hardware while the genes are thesoftware. In this sense, the purpose of gene therapy is to provide thecell with a new program (genetic information) so as to reprogram adysfunctional cell to perform a normal function. The addition of a newcellular function is provided by the insertion of a foreign gene thatexpresses a foreign protein or a native protein at amounts that are notpresent in the patient.

The inhibition of a cellular function is provided by anti-senseapproaches (that is acting against messenger RNA) and that includesoligonucleotides complementary to the messenger RNA sequence andribozymes. Messenger RNA (mRNA) is an intermediate in the expression ofthe DNA gene. The mRNA is translated into a protein. “Anti-sense”methods use a RNA sequence or an oligonucleotide that is madecomplementary to the target mRNA sequence and therefore bindsspecifically to the target messenger RNA. When this anti-sense sequencebinds to the target mRNA, the mRNA is somehow destroyed or blocked frombeing translated. Ribozymes destroy a specific mRNA by a differentmechanism. Ribozymes are RNA's that contain sequence complementary tothe target messenger RNA plus a RNA sequence that acts as an enzyme tocleave the messenger RNA, thus destroying it and preventing it frombeing translated. When these anti-sense or ribozyme sequences areintroduced into a cell, they would inactivate their specific target mRNAand reduce their disease-causing properties.

Several recessive genetic disorders are being considered for genetherapy. One of the first uses of gene therapy in humans has been usedfor the genetic deficiency of the adenosine deaminase (ADA) gene. Otherclinical gene therapy trials have been conducted for cystic fibrosis,familial hypercholesteremia caused by a defective LDL-receptor gene andpartial ornithine transcarbomylase deficiency. Both indirect and directgene therapy approaches are being developed for Duchenne musculardystrophy. Patients with this type of muscular dystrophy eventually diefrom loss of their respiratory muscles. Direct approaches include theintramuscular injection of naked plasmid DNA or adenoviral vectors.

A wide variety of gene therapy approaches for cancer are underinvestigation in animals and in human clinical trials. One approach isto express in lymphocytes and in the tumor cells, cytokine genes thatstimulate the immune system to destroy the cancer cells. The cytokinegenes would be transferred into the lymphocytes by removing thelymphocytes from the body and infecting them with a retroviral vectorcarrying the cytokine gene. The tumor cells would be similarlygenetically modified by this indirect approach to express cytokineswithin the tumor. Direct approaches involving the expression ofcytokines in tumor cells in situ are also being considered. Other genesbesides cytokines may be able to induce an immune response against thecancer. One approach that has entered clinical trials is the directinjection of HLA-B7 gene (which encodes a potent immunogen) within lipidvesicles into malignant melanomas in order to induce a more effectiveimmune response against the cancer.

“Suicide” genes are genes that kill cells that express the gene. Forexample, the diphtheria toxin gene directly kills cells. The Herpesthymidine kinase (TK) gene kills cells in conjunction with acyclovir (adrug used to treat Herpes viral infections). Other gene therapyapproaches take advantage of our knowledge of oncogenes and suppressortumor genes—also known as anti-oncogenes. The loss of a functioninganti-oncogene plays a decisive role in childhood tumors such asretinoblastoma, osteosarcoma and Wilms tumor and may play an importantrole in more common tumors such as lung, colon and breast cancer.Introduction of the normal anti-oncogene back into these tumor cells mayconvert them back to normal cells. The activation of oncogenes alsoplays an important role in the development of cancers. Since theseoncogenes operate in a “dominant” fashion, treatment will requireinactivation of the abnormal oncogene. This can be done using either“anti-sense” or ribozyme methods that selectively inactivate a specificmessenger RNA in a cell.

Gene therapy can be used as a type of vaccination to prevent infectiousdiseases and cancer. When a foreign gene is transferred into a cell andthe protein is made, the foreign protein is presented to the immunesystem differently from simply injecting the foreign protein into thebody. This different presentation is more likely to cause acell-mediated immune response which is important for fighting latentviral infections such as human immunodeficiency virus (HIV causes AIDS),Herpes and cytomegalovirus. Expression of the viral gene within a cellsimulates a viral infection and induces a more effective immune responseby fooling the body that the cell is actually infected by the virus,without the danger of an actual viral infection.

One direct approach uses the direct intramuscular injection of nakedplasmid DNA to express a viral gene in muscle cells. The “gun” has alsobeen shown to be effective at inducing an immune response by expressingforeign genes in the skin. Other direct approaches involving the use ofretroviral, vaccinia or adenoviral vectors are also being developed. Anindirect approach has been developed to remove fibroblasts from theskin, infect them with a retroviral vector carrying a viral gene andtransplant the cells back into the body. The envelope gene from the AIDSvirus (HIV) is often used for these purposes. Many cancer cells expressspecific genes that normal cells do not. Therefore, these genesspecifically expressed in cancer cells can be used for immunizationagainst cancer.

Besides the above immunization approaches, several other gene therapiesare being developed for treating infectious disease. Most of these newapproaches are being developed for HIV infection and AIDS. Many of themwill involve the delivery of anti-sense or ribozyme sequences directedagainst the particular viral messenger RNA. These anti-sense or ribozymesequences will block the expression of specific viral genes and abortthe viral infection without damaging the infected cell. Another approachsomewhat similar to the ant-sense approaches is to overexpress thetarget sequences for these regulatory HIV sequences.

Gene therapy efforts would be directed at lowering the risk factorsassociated with atherosclerosis. Overexpression of the LDL receptor genewould lower blood cholesterol in patients not only with familialhypercholesteremia but with other causes of high cholesterol levels. Thegenes encoding the proteins for HDL (“the good cholesterol”) could beexpressed also in various tissues. This would raise HDL levels andprevent atherosclerosis and heart attacks. Tissue plasminogen activator(tPA) protein is being given to patients immediately after theirmyocardial infarction to digest the blood clots and open up the blockedcoronary blood vessels. The gene for tPA could be expressed in theendothelial cells lining the coronary blood vessels and thereby deliverthe tPA locally without providing tPA throughout the body. Anotherapproach for coronary vessel disease is to express a gene in the heartthat produces a protein that causes new blood vessels to grow. Thiswould increase collateral blood flow and prevent a myocardial infarctionfrom occurring.

Neurodegenerative disorders such as Parkinson's and Alzheimer's diseasesare good candidates for early attempts at gene therapy. Arthritis couldalso be treated by gene therapy. Several proteins and their genes (suchas the IL-1 receptor antagonist protein) have recently been discoveredto be anti-inflammatory. Expression of these genes in joint (synovial)fluid would decrease the joint inflammation and treat the arthritis.

In addition, methods are being developed to directly modify the sequenceof target genes and chromosomal DNA. The delivery of a nucleic acid orother compound that modifies the genetic instruction (e.g., byhomologous recombination) can correct a mutated gene or mutate afunctioning gene.

Polymers for Drug and Nucleic Acid Delivery

Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used in research for the delivery ofnucleic acids (polynucleotides and oligonucleotides) to cells with aneventual goal of providing therapeutic processes. Such processes havebeen termed gene therapy or anti-sense therapy. One of the severalmethods of nucleic acid delivery to the cells is the use ofDNA-polycation complexes. It has been shown that cationic proteins likehistones and protamines or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular delivery agents whilesmall polycations like spermine are ineffective. The following are someimportant principles involving the mechanism by which polycationsfacilitate uptake of DNA:

Polycations provide attachment of DNA to the cell surface. The polymerforms a cross-bridge between the polyanionic nucleic acids and thepolyanionic surfaces of the cells. As a result the main mechanism of DNAtranslocation to the intracellular space might be non-specificadsorptive endocytosis which may be more effective then liquidendocytosis or receptor-mediated endocytosis. Furthermore, polycationsare a convenient linker for attaching specific ligands to DNA and asresult, DNA-polycation complexes can be targeted to specific cell types.

Polycations protect DNA in complexes against nuclease degradation. Thisis important for both extra—and intracellular preservation of DNA. Geneexpression is also enabled or increased by preventing endosomeacidification with NH₄Cl or chloroquine. Polyethylenimine, whichfacilitates gene expression without additional treatments, probablydisrupts endosomal function itself. Disruption of endosomal function hasalso been accomplished by linking to the polycation endosomal-disruptiveagents such as fusion peptides or adenoviruses.

Polycations can also facilitate DNA condensation. The volume which oneDNA molecule occupies in a complex with polycations is drastically lowerthan the volume of a free DNA molecule. The size of a DNA/polymercomplex is probably critical for gene delivery in vivo. In terms ofintravenous injection, DNA needs to cross the endothelial barrier andreach the parenchymal cells of interest. The largest endotheliafenestrae (holes in the endothelial barrier) occur in the liver and havean average diameter of 100 nm. The trans-epithelial pores in otherorgans are much smaller, for example, muscle endothelium can bedescribed as a structure which has a large number of small pores with aradius of 4 nm, and a very low number of large pores with a radius of20–30 nm. The size of the DNA complexes is also important for thecellular uptake process. After binding to the cells the DNA-polycationcomplex should be taken up by endocytosis. Since the endocytic vesicleshave a homogenous internal diameter of about 100 nm in hepatocytes andare of similar size in other cell types, DNA complexes smaller than 100nm are preferred.

Condensation of DNA

A significant number of multivalent cations with widely differentmolecular structures have been shown to induce condensation of DNA.

Two approaches for compacting (used herein as an equivalent to the termcondensing) DNA:

1. Multivalent cations with a charge of three or higher have been shownto condense DNA. These include spermidine, spermine, Co(NH₃)₆ ³⁺,Fe³⁺,and natural or synthetic polymers such as histone H1, protamine,polylysine, and polyethylenimine. Analysis has shown DNA condensation tobe favored when 90% or more of the charges along the sugar-phosphatebackbone are neutralized.

2. Polymers (neutral or anionic) which can increase repulsion betweenDNA and its surroundings have been shown to compact DNA. Mostsignificantly, spontaneous DNA self-assembly and aggregation processhave been shown to result from the confinement of large amounts of DNA,due to excluded volume effect.

Depending upon the concentration of DNA, condensation leads to threemain types of structures:

1) In extremely dilute solution (about 1 μg/mL or below), long DNAmolecules can undergo a monomolecular collapse and form structuresdescribed as toroid.

2) In very dilute solution (about 10 μg/mL) microaggregates form withshort or long molecules and remain in suspension. Toroids, rods andsmall aggregates can be seen in such solution.

3) In dilute solution (about 1 mg/mL) large aggregates are formed thatsediment readily.

Toroids have been considered an attractive form for gene deliverybecause they have the smallest size. While the size of DNA toroidsproduced within single preparations has been shown to vary considerably,toroid size is unaffected by the length of DNA being condensed. DNAmolecules from 400 bp to genomic length produce toroids similar in size.Therefore one toroid can include from one to several DNA molecules. Thekinetics of DNA collapse by polycations that resulted in toroids is veryslow. For example DNA condensation by Co(NH₃)₆Cl₃ needs 2 hours at roomtemperature.

The mechanism of DNA condensation is not clear. The electrostatic forcebetween unperturbed helices arises primarily from a counterionfluctuation mechanism requiring multivalent cations and plays a majorrole in DNA condensation. The hydration forces predominate overelectrostatic forces when the DNA helices approach closer then a fewwater diameters. In a case of DNA-polymeric polycation interactions, DNAcondensation is a more complicated process than the case of lowmolecular weight polycations. Different polycationic proteins cangenerate toroid and rod formation with different size DNA at a ratio ofpositive to negative charge of two to five. T4 DNA complexes withpolyarginine or histone can form two types of structures; an elongatedstructure with a long axis length of about 350 nm (like free DNA) anddense spherical particles. Both forms exist simultaneously in the samesolution. The reason for the co-existence of the two forms can beexplained as an uneven distribution of the polycation chains among theDNA molecules. The uneven distribution generates two thermodynamicallyfavorable conformations.

The electrophoretic mobility of DNA-polycation complexes can change fromnegative to positive in excess of polycation. It is likely that largepolycations don't completely align along DNA but form polymer loops thatinteract with other DNA molecules. The rapid aggregation and strongintermolecular forces between different DNA molecules may prevent theslow adjustment between helices needed to form tightly packed orderlyparticles.

As previously stated, preparation of polycation-condensed DNA particlesis of particular importance for gene therapy, more specifically,particle delivery such as the design of non-viral gene transfer vectors.Optimal transfection activity in vitro and in vivo can require an excessof polycation molecules. However, the presence of a large excess ofpolycations may be toxic to cells and tissues. Moreover, thenon-specific binding of cationic particles to all cells forestallscellular targeting. Positive charge also has an adverse influence onbiodistribution of the complexes in vivo.

Several modifications of DNA-cation particles have been created tocircumvent the nonspecific interactions of the DNA-cation particle andthe toxicity of cationic particles. Examples of these modificationsinclude attachment of steric stabilizers, e.g. polyethylene glycol,which inhibit nonspecific interactions between the cation and biologicalpolyanions. Another example is recharging the DNA particle by theadditions of polyanions which interact with the cationic particle,thereby lowering its surface charge, i.e. recharging of the DNA particleU.S. Ser. No. 09/328,975. Another example is cross-linking the polymersand thereby caging the complex U.S. Ser. No. 08/778,657, now U.S. Pat.No. 6,126,964, U.S. Ser. No. 09/000,692, now U.S. Pat. No. 6,339,067,U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070299, abandoned, and U.S.Ser. No. 09/464,871. Nucleic acid particles can be formed by theformation of chemical bonds and template polymerization U.S. Ser. No.08/778,657, now U.S. Pat. No. 6,126,964, U.S. Ser. No. 09/000,692, nowU.S. Pat. No. 6,339,067, U.S. Ser. No. 97/24089, U.S. Ser. No.09/070299, abandoned, and U.S. Ser. No. 09/464,871, abandoned.

A problem with these modifications is that they are most likelyirreversible rendering the particle unable to interact with the cell tobe transfected, and/or incapable of escaping from the lysosome oncetaken into a cell, and/or incapable of entering the nucleus once insidethe cell. A method for formation of DNA particles that is reversibleunder conditions found in the cell may allow for effective delivery ofDNA. The conditions that cause the reversal of particle formation maybe, but not limited to, the pH, ionic strength, oxidative or reductiveconditions or agents, or enzymatic activity.

DNA Template Polymerization

Low molecular weight cations with valency, i.e. charge, <+3 fail tocondense DNA in aqueous solutions under normal conditions. However,cationic molecules with the charge <+3 can be polymerized in thepresence of DNA and the resulting polymers can cause DNA to condenseinto compact structures. Such an approach is known in synthetic polymerchemistry as template polymerization. During this process, monomers(which are initially weakly associated with the template) are positionedalong template's backbone, thereby promoting their polymerization. Weakelectrostatic association of the nascent polymer and the templatebecomes stronger with chain growth of the polymer. Trubetskoy et al usedtwo types of polymerization reactions to achieve DNA condensation: steppolymerization and chain polymerization (V S Trubetskoy, V G Budker, L JHanson, P M Slattum, J A Wolff, L E Hagstrom. Nucleic Acids Res.26:4178–4185, 1998) U.S. Ser. No. 08/778,657, now U.S. Pat. No.6,126,964, U.S. Ser. No. 09/000,692, now U.S. Pat. No. 6,339,067, U.S.Ser. No. 97/24089, U.S. Ser. No. 09/070299, abandoned, and U.S. Ser. No.09/464,871, abandoned. Bis(2-aminoethyl)-1,3-propanediamine (AEPD), atetramine with 2.5 positive charges per molecule at pH 8 was polymerizedin the presence of plasmid DNA using cleavable disulfide amino-reactivecross-linkers dithiobis (succinimidyl propionate) anddimethyl-3,3′-dithiobispropionimidate. Both reactions yieldedDNA/polymer complexes with significant retardation in agaroseelectrophoresis gels demonstrating significant binding and DNAcondensation. Treatment of the polymerized complexes with 100 mMdithiothreitol (DTT) resulted in the pDNA returning to its normalsupercoiled position following electrophoresis proving thus cleavage thebackbone of the. The template dependent polymerization process was alsotested using a 14 mer peptide encoding the nuclear localizing signal(NLS) of SV40 T antigen (SEQ ID NO: 1) as a cationic “macromonomer”.Other studies included pegylated comonomer (PEG-AEPD) into the reactionmixture and resulted in “worm”-like structures (as judged bytransmission electron microscopy) that have previously been observedwith DNA complexes formed from block co-polymers of polylysine and PEG(M A Wolfert, E H Schacht, V Toncheva, K Ulbrich, O Nazarova, L WSeymour. Human Gene Ther. 7:2123–2133, 1996). Blessing et al usedbisthiol derivative of spermine and reaction of thiol-disulfide exchangeto promote chain growth. The presence of DNA accelerated thepolymerization reaction as measured the rate of disappearance of freethiols in the reaction mixture (T Blessing, J S Remy, J P Behr. J. Am.Chem. Soc. 120:8519–8520, 1998).

“Caging” of Polycation-Condensed DNA Particles.

The stability of DNA nanoassemblies based on DNA condensation isgenerally low in vivo because they easily engage in polyion exchangereactions with strong polyanions. The process of exchange consists oftwo stages: 1) rapid formation of a triple DNA-polycation-polyanioncomplex, 2) slow substitution of one same-charge polyion with another.At equilibrium conditions, the whole process eventually results information of a new binary complex and an excess of a third polyion. Thepresence of low molecular weight salt can greatly accelerate suchexchange reactions, which often result in complete disassembly ofcondensed DNA particles. Hence, it is desirable to obtain morecolloidally stable structures where DNA would stay in its condensed formin complex with corresponding polycation independently of environmentconditions.

The complete DNA condensation upon neutralization of only 90% of thepolymer's phosphates results in the presence of unpaired positivecharges in the DNA particles. If the polycation contains such reactivegroups, such as primary amines, these unpaired positive charges may bemodified. This modification allows practically limitless possibilitiesof modulating colloidal properties of DNA particles via chemicalmodifications of the complex. We have demonstrated the utility of suchreactions using traditional DNA-poly-L-lysine (DNA/PLL) system reactedwith the cleavable cross-linking reagentdimethyl-3,3′-dithiobispropionimidate (DTBP) which reacts with primaryamino groups with formation of amidines (V S Trubetskoy, A Loomis, P MSlattum, J E Hagstrom, V G Budker, J A Wolff. Bioconjugate Chem.10:624–628, 1999) U.S. Ser. No. 08/778,657, now U.S. Pat. No. 6,126,964,U.S. Ser. No. 09/000,692, now U.S. Pat. No. 6,339,067, U.S. Ser. No.97/24089, U.S. Ser. No. 09/070299, abandoned, and U.S. Ser. No.09/464,871, abandoned. Similar results were achieved with otherpolycations including poly(allylamine) and histone H1. The use ofanother bifucntional reagent, glutaraldehyde, has been described forstabilization of DNA complexes with cationic peptide CWK18 (R C Adam, KG Rice. J. Pharm. Sci. 739–746, 1999).

Recharging.

The caging approach described above could lead to more colloidallystable DNA assemblies. However, this approach may not change theparticle surface charge. Caging with bifunctional reagents, whichpreserve positive charge of amino group, keeps the particle positive.However, negative surface charge would be more desirable for manypractical applications, i.e. in vivo delivery. The phenomenon of surfacerecharging is well known in colloid chemistry and is described in greatdetail for lyophobic/lyophilic systems (for example, silver halidehydrosols). Addition of polyion to a suspension of latex particles withoppositely-charged surface leads to the permanent absorption of thispolyion on the surface and, upon reaching appropriate stoichiometry,changing the surface charge to opposite one. This whole process is saltdependent with flocculation to occur upon reaching the neutralizationpoint.

We have demonstrated that similar layering of polyelectrolytes can beachieved on the surface of DNA/polycation particles (V S Trubetskoy, ALoomis, J E Hagstrom, V G Budker, J A Wolff. Nucleic Acids Res.27:3090–3095, 1999). The principal DNA-polycation (DNA/pC) complex usedin this study was DNA/PLL (1:3 charge ratio) formed in low salt 25 mMHEPES buffer and recharged with increasing amounts of variouspolyanions. The DNA particles were characterized after addition of athird polyion component to a DNA/polycation complex using a new DNAcondensation assay (V S Trubetskoy, P M Slattum, J E Hagstrom, J AWolff, V G Budker. Anal. Biochem. 267:309–313, 1999) and static lightscattering. It has been found that certain polyanions such aspoly(methacrylic acid) and poly(aspartic acid) decondensed DNA inDNA/PLL complexes. Surprisingly, polyanions of lower charge density suchas succinylated PLL and poly(glutamic acid), even when added in 20-foldcharge excess to condensing polycation (PLL) did not decondense DNA inDNA/PLL (1:3) complexes. Further studies have found that displacementeffects are salt-dependent. In addition, poly-L-glutamic acid but notthe relatively weaker polyanion succinylated poly-L-lysine (SPLL)displaces DNA at higher sodium chloride concentrations. Measurement ofζ-potential of DNA/PLL particles during titration with SPLL revealed thechange of particle surface charge at approximately the chargeequivalency point. Thus, it can be concluded that addition of low chargedensity polyanion to the cationic DNA/PLL particles results in particlesurface charge reversal while maintaining condensed DNA core intact.Finally, DNA/polycation complexes can be both recharged and crosslinkedor caged U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser.No. 97/24089, U.S. Ser. No. 09/070299, and U.S. Ser. No. 09/464,871.

The Use of pH-Sensitive Lipids, Amphipathic Compounds, and Liposomes forDrug and Nucleic Acid Delivery

After the landmark description of DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)[Felgner, P L, Gadek, T R, Holm, M, et al. Lipofection: a highlyefficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad.Sci. USA. 1987;84:7413–7417], a plethora of cationic lipids have beensynthesized. Basically, all the cationic lipids are amphipathiccompounds that contain a hydrophobic domain, a spacer, andpositively-charged amine. The hydrophobic domains are typicallyhydrocarbon chains such as fatty acids derived from oleic or myristicacid. The hydrocarbon chains are often joined either by ether or esterbonds to a spacer such as glycerol. Quaternary amines often compose thecationic groups. Usually, the cationic lipids are mixed with a fusogeniclipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to formliposomes. The mixtures are mixed in chloroform that is then dried.Water is added to the dried lipid film and unilamellar liposomes formduring sonication. Multilamellar cationic liposomes and cationicliposomes/DNA complexes prepared by the reverse-phase evaporation methodhave also been used for transfection. Cationic liposomes have also beenprepared by an ethanol injection technique.

Several cationic lipids contain a spermine group for binding to DNA.DOSPA, the cationic lipid within the LipofectAMINE formulation (LifeTechnologies) contains a spermine linked via a amide bond and ethylgroup to a trimethyl, quaternary amine [Hawley-Nelson, P, Ciccarone, Vand Jessee, J. Lipofectamine reagent: A new, higher efficiencypolycationic liposome transfection reagent. Focus 1993;15:73–79]. AFrench group has synthesized a series of cationic lipids such as DOGS(dioctadecylglycinespermine) that contain spermine [Remy, J-S, Sirlin,C, Vierling, P, et al. Gene transfer with a series of lipophilicDNA-binding molecules. Bioconjugate Chem. 1994;5:647–654]. DNA has alsobeen transfected by lipophilic polylysines which containdipalmotoylsuccinylglycerol chemically-bonded to low molecular weight(˜3000 MW) polylysine [Zhou, X, Kilbanov, A and Huang, L. Lipophilicpolylysines mediate efficient DNA transfection in mammalian cells.Biochim. Biophys. Acta 1991;1065:8–14. Zhou, X and Huang, L. DNAtransfection mediated by cationic liposomes containing lipopolylysine:Characterization and mechanism of action. Biochim. Biophys. Acta 1994;1195–203].

Other studies have used adjuvants with the cationic liposomes.Transfection efficiency into Cos cells was increased when amphiphilicpeptides derived from influenza virus hemagglutinin were added toDOTMA/DOPE liposomes [Kamata, H, Yagisawa, H, Takahashi, S, et al.Amphiphilic peptides enhance the efficiency of liposome-mediated DNAtransfection. Nucleic Acids Res. 1994;22:536–537]. Cationic lipids havebeen combined with galactose ligands for targeting to the hepatocyteasialoglycoprotein receptor [Remy, J-S, Kichler, A, Mordvinov, V, et al.Targeted gene transfer into hepatoma cells with lipopolyamine-condensedDNA particles presenting galactose ligands: A stage toward artificialviruses. Proc. Natl. Acad. Sci. USA 1995;92:1744–1748]. Thiol-reactivephospholipids have also been incorporated into cationic lipid/pDNAcomplexes to enable cellular binding even when the net charge of thecomplex is not positive [Kichler, A, Remy, J-S, Boussif, O, et al.Efficient gene delivery with neutral complexes of lipospermine andthiol-reactive phospholipids. Biochem. Biophys. Res. Comm.1995;209:444–450]. DNA-dependent template process convertedthiol-containing detergent possessing high critical micelleconcentration into dimeric lipid-like molecule with apparently low watersolubility.

Cationic liposomes may deliver DNA either directly across the plasmamembrane or via the endosome compartment. Regardless of its exact entrypoint, much of the DNA within cationic liposomes does accumulate in theendosome compartment. Several approaches have been investigated toprevent loss of the foreign DNA in the endosomal compartment byprotecting it from hydrolytic digestion within the endosomes or enablingits escape from endosomes into the cytoplasm. They include the use ofacidotropic (lysomotrophic), weak amines such as chloroquine thatpresumably prevent DNA degradation by inhibiting endosomal acidification[Legendre, J. & Szoka, F. Delivery of plasmid DNA into mammalian celllines using pH-sensitive liposomes: Comparison with cationic liposomes.Pharmaceut. Res. 9, 1235–1242 (1992)]. Viral fusion peptides or wholevirus have been included to disrupt endosomes or promote fusion ofliposomes with endosomes, and facilitate release of DNA into thecytoplasm [Kamata, H., Yagisawa, H., Takahashi, S. & Hirata, H.Amphiphilic peptides enhance the efficiency of liposome-mediated DNAtransfection. Nucleic Acids Res. 22, 536–537 (1994). Wagner, E., Curiel,D. & Cotten, M. Delivery of drugs, proteins and genes into cells usingtransferrin as a ligand for receptor-mediated endocytosis. Advanced DrugDelivery Reviews 14,113–135 (1994)].

Knowledge of lipid phases and membrane fusion has been used to designpotentially more versatile liposomes that exploit the endosomalacidification to promote fusion with endosomal membranes. Such anapproach is best exemplified by anionic, pH-sensitive liposomes thathave been designed to destabilize or fuse with the endosome membrane atacidic pH [Duzgunes, N., Straubinger, R. M., Baldwin, P. A. &Papahadjopoulos, D. PH-sensitive liposomes. (eds Wilschub, J. &Hoekstra, D.) p. 713–730 (Marcel Deker INC, 1991)]. All of the anionic,pH-sensitive liposomes have utilized phosphatidylethanolamine (PE)bilayers that are stabilized at non-acidic pH by the addition of lipidsthat contain a carboxylic acid group. Liposomes containing only PE areprone to the inverted hexagonal phase (H_(II)). In pH-sensitive, anionicliposomes, the carboxylic acid's negative charge increases the size ofthe lipid head group at pH greater than the carboxylic acid's pK_(a) andthereby stabilizes the phosphatidylethanolamine bilayer. At acidic pHconditions found within endosomes, the uncharged or reduced chargespecies is unable to stabilize the phosphatidylethanolamine-richbilayer. Anionic, pH-sensitive liposomes have delivered a variety ofmembrane-impermeable compounds including DNA. However, the negativecharge of these pH-sensitive liposomes prevents them from efficientlytaking up DNA and interacting with cells; thus decreasing their utilityfor transfection. We have described the use of cationic, pH-sensitiveliposomes to mediate the efficient transfer of DNA into a variety ofcells in culture U.S. Ser. No. 08/530,598, now U.S. Pat. No. 5,744,335,and U.S. Ser. No. 09/020,566, now U.S. Pat. No. 6,180,784.

The Use of pH-Sensitive Polymers for Drug and Nucleic Acid Delivery

Polymers that pH-sensitive are have found broad application in the areaof drug delivery exploiting various physiological and intracellular pHgradients for the purpose of controlled release of drugs (both lowmolecular weight and polymeric). pH sensitivity can be broadly definedas any change in polymer's physico-chemical properties over certainrange of pH. More narrow definition demands significant changes in thepolymer's ability to retain (release) a bioactive substance (drug) in aphysiologically tolerated pH range (usually pH 5.5–8). pH-sensitivitypresumes the presence of ionizable groups in the polymer (polyion). Allpolyions can be divided into three categories based on their ability todonate or accept protons in aqueous solutions: polyacids, polybases andpolyampholytes. Use of pH-sensitive polyacids in drug deliveryapplications usually relies on their ability to become soluble with thepH increase (acid/salt conversion), to form complex with other polymersover change of pH or undergo significant change inhydrophobicity/hydrophilicity balance. Combinations of all three abovefactors are also possible.

Copolymers of polymethacrylic acid (Eudragit S, Rohm America) are knownas polymers which are insoluble at lower pH but readily solubilized athigher pH, so they are used as enteric coatings designed to dissolve athigher intestinal pH (Z Hu et al. J. Drug Target., 7, 223, 1999). Atypical example of pH-dependent complexation is copolymers ofpolyacrylate(graft)ethyleneglycol which can be formulated into variouspH-sensitive hydrogels which exhibit pH-dependent swelling and drugrelease (F Madsen et al., Biomaterials, 20, 1701, 1999).Hydrophobically-modified N-isopropylacrylamide-methacrylic acidcopolymer can render regular egg PC liposomes pH-sensitive bypH-dependent interaction of grafted aliphatic chains with lipid bilayer(O Meyer et al., FEBS Lett., 421, 61, 1998). Polymers with pH-mediatedhydrophobicity (like polyethylacrylic acid) can be used as endosomaldisrupters for cytoplasmic drug delivery (Murthy, N., Robichaud, J. R.,Tirrell, D. A., Stayton, P. S., Hoffman, A. S. J. Controlled Release 61,137, 1999).

Polybases have found broad applications as agents for nucleic aciddelivery in transfection/gene therapy applications due to the fact theyare readily interact with polyacids. A typical example ispolyethyleneimine (PEI). This polymer secures nucleic acid electrostaticadsorption on the cell surface followed by endocytosis of the wholecomplex. Cytoplasmic release of the nucleic acid occurs presumably viathe so called “proton sponge” effect according to which pH-sensitivityof PEI is responsible for endosome rupture due to osmotic swellingduring its acidification (O Boussif et al. Proc. Natl. Acad. Sci. USA92, 7297, 1995). Cationic acrylates possess the similar activity (forexample, poly-((2-dimethylamino)ethyl methacrylate) (P van de Weteringet al. J. Controlled Release 64, 193, 2000). However, polybases due totheir polycationic nature pH-sensitive polybases have not found broad invivo application so far, due to their acute systemic toxicity in vivo (JH Senior, Biochim. Biophys. Acta, 1070, 173, 1991). Milder polybases(for example, linear PEI) are better tolerated and can be usedsystemically for in vivo gene transfer (D Goula et al. Gene Therapy 5,712, 1998).

Membrane Active Compounds

Many biologically active compounds, in particular large and/or chargedcompounds, are incapable of crossing biological membranes. In order forthese compounds to enter cells, the cells must either take them up byendocytosis, into endosomes, or there must be a disruption of thecellular membrane to allow the compound to cross. In the case ofendosomal entry, the endosomal membrane must be disrupted to allow forthe entrance of the compound in the interior of the cell. Therefore,either entry pathway into the cell requires a disruption of the cellularmembrane. There exist compounds termed membrane active compounds thatdisrupt membranes. One can imagine that if the membrane active agentwere operative in a certain time and place it would facilitate thetransport of the biologically active compound across the biologicalmembrane. The control of when and where the membrane active compound isactive is crucial to effective transport. If the membrane activecompound is too active or active at the wrong time, then no transportoccurs or transport is associated with cell rupture and thereby celldeath. Nature has evolved various strategies to allow for membranetransport of biologically active compounds including membrane fusion andthe use membrane active compounds whose activity is modulated such thatactivity assists transport without toxicity. Many lipid-based transportformulations rely on membrane fusion and some membrane active peptides'activities are modulated by pH. In particular, viral coat proteins areoften pH-sensitive, inactive at neutral or basic pH and active under theacidic conditions found in the endosome.

Small Molecular Endosomolytic Agents

A cellular transport step that has attracted attention for gene transferis that of DNA release from intracellular compartments such as endosomes(early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum,golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum.Release includes movement out of an intracellular compartment intocytoplasm or into an organelle such as the nucleus. A number ofchemicals such as chloroquine, bafilomycin or Brefeldin A1 have beenused to disrupt or modify the trafficking of molecules throughintracellular pathways. Chloroquine decreases the acidification of theendosomal and lysosomal compartments but also affects other cellularfunctions. Brefeldin A, an isoprenoid fungal metabolite, collapsesreversibly the Golgi apparatus into the endoplasmic reticulum and theearly endosomal compartment into the trans-Golgi network (TGN) to formtubules. Bafilomycin A₁, a macrolide antibiotic is a more specificinhibitor of endosomal acidification and vacuolar type H⁺-ATPase thanchloroquine.

Viruses, Proteins and Peptides for Disruption of Endosomes and EndosomalFunction

Viruses such as adenovirus have been used to induce gene release fromendosomes or other intracellular compartments (D. Curiel, Agarwal, S.,Wagner, E., and Cotten, M. PNAS 88:8850, 1991). Rhinovirus has also beenused for this purpose (W. Zauner et al. J. Virology 69:1085–92, 1995).Viral components such as influenza virus hemagglutinin subunit HA-2analogs has also been used to induce endosomal release (E. Wagner et al.PNAS 89:7934, 1992). Amphipathic peptides resembling the N-terminal HA-2sequence has been studied (K. Mechtler and E. Wagner, New J. Chem.21:105–111, 1997). Parts of the pseudonmonas exotoxin and diptheriatoxin have also been used for drug delivery (I. Pastan and D.FitzGerald. J. Biol. Chem. 264:15157, 1989).

A variety of synthetic amphipathic peptides have been used to enhancetransfection of genes (N. Ohmori et al. Biochem. Biophys. Res. Commun.235:726, 1997). The ER-retaining signal (KDEL sequence) has beenproposed to enhance delivery to the endoplasmic reticulum and preventdelivery to lysosomes (S. Seetharam et al. J. Biol. Chem. 266:17376,1991).

The present invention provides for a new group of membrane activecompounds that can enhance the delivery of nucleic acids.

Other Cellular and Intracellular Gradients Useful for Delivery

Nucleic acid and gene delivery may involve the biological pH gradientthat is active within organisms as a factor in delivering apolynucleotide to a cell. Different pathways that may be affected by thepH gradient include cellular transport mechanisms, endosomaldisruption/breakdown, and particle disassembly (release of the DNA).Other gradients that can be useful in gene therapy research involveionic gradients that are related to cells. For example, both Na⁺ and K⁺have large concentration gradients that exist across the cell membrane.Systems containing metal-binding groups can utilize such gradients toinfluence delivery of a polynucleotide to a cell. Changes in the osmoticpressure in the endosome also have been used to disrupt membranes andallow for transport across membrane layer. Buffering of the endosome pHmay cause these changes in osmotic pressure. For example, the “protonsponge” effect of PEI (O Boussif et al. Proc. Natl. Acad. Sci. USA 92,7297, 1995) and certain polyanions (Murthy, N., Robichaud, J. R.,Tirrell, D. A., Stayton, P. S., Hoffman, A. S. Journal of ControlledRelease 1999, 61, 137) are postulated to cause an increase in the ionicstrength inside of the endosome, which causes a increase in osmoticpressure. This pressure increase results in membrane disruption andrelease of the contents of the endosome.

In addition to pH and other ionic gradients, there exist otherdifference in the chemical environment associated with cellularactivities that may be used in gene delivery. In particular enzymaticactivity both extra and intracellularly may be used to deliver the geneof interest either by aiding in the delivery to the cell or escape fromintracellular compartments. Proteases, found in serum, lysosome andcytoplasm, may be used to disrupt the particle and allow its interactionwith the cell surface or cause it fracture the intracellularcompartment, e.g. endosome or lysosome, allowing the gene to be releasedintracellularly.

SUMMARY

Compounds and methods are described for enhancing the delivery ofbiologically active compounds including peptides, small molecular drugsand nucleic acids. Novel pH-labile and membrane active compounds aredescribed. Some of these compounds are cleaved at acidic pH; therebyincreasing their membrane activity. Some of these novel compounds alsohave use as detergents.

DETAILED DESCRIPTION

The present invention relates to the delivery of desired compounds(e.g., drugs and nucleic acids) into cells using pH-labile polymers andmembrane active compounds coupled with labile compounds. The presentinvention provides compositions and methods for delivery and release ofa compound of interest to a cell.

Noncovalent molecule-molecule interactions, which are the basis ofDNA-polycation particle formation, rely on discreet interactions betweenthe functional groups on the interacting molecules. It is quite apparentthat if one modifies the interacting functional groups, one changes thewhole molecule-molecule interaction. This is true for small moleculesand large macromolecules. For example methyl alcohol is a liquid capableof hydrogen bonding with water, which confers the compound with watersolubility. In contrast, conversion of the alcohol functional group to amethyl ether to form dimethyl ether renders the molecule in to a waterinsoluble gas. Many other examples may be observed in small molecularweight drug-receptor interactions. DNA interacts with the polycationpoly-L-lysine to form condensed DNA particles. If the amino groups ofpoly-L-lysine are converted to carboxylate groups as in succinylatedpoly-L-lysine there is no interaction with the polyanion DNA. Theidentities of the functional groups on a molecule dictate itsinteractions with other molecules. Therefore, the ability to control theidentity of the function groups on a molecule allows one to control itsinteractions. As a consequence, controlled and reversible functionalgroup modification is important if one want to modulate a molecule'sinteractions. This control is of particular importance when the moleculein question is biologically active. For example, one may not want toadminister cytotoxic drugs directly. In this case, one may administer aprodrug that is itself inactive, but becomes active by change(s) infunctional group(s) after delivery.

Prior to the present invention, delivery systems suffered from slowreversibility—or irreversibility—and/or high toxicity. For example, manycationic polymers such as poly-L-lysine (PLL) and polyethylenimine (PEI)form positively charged condensed particles with DNA. In vitro, theseparticles are relatively good reagents, compared to DNA alone, for thetransfer of DNA into cells. However, these particles are poor transferreagents in vivo due to their toxicity and relatively stable interactionwith DNA, which renders their complexation irreversible underphysiological conditions. There are several barriers that thesecomplexes must overcome for them to be efficient gene transfer reagents:stable enough to protect the DNA from nucleases and aid in delivery tothe cell, yet the DNA polycation complex must be disrupted—therebyallowing transcription to occur. Additionally, if the complex is takeninto the cell through the process of endocytosis, the complex mustescape the endosome before being taken into the lysosome and beingdigested.

To increase the stability of DNA particles in serum, certain embodimentsof the present invention provide polyanions that form a third layer inthe DNA-polycation complex and it is negatively charged. To assist inthe disruption of the DNA complexes, certain embodiments of the presentinvention provide synthesized polymers that are cleaved in the acidconditions found in the endosome (i.e., pH 5–7). For example, thepresent invention provides for the cleavage or alteration of a labilechemical group once the complex is in the desired environment: cleavageof the polymer backbone resulting in smaller polyions or cleavage of thelink between the polymer backbone and the ion resulting in an ion and anpolymer. In either case, the number of molecules in the endosomeincreases. This alteration may facilitate the release of the deliveredcompound into the cytoplasm. Although it is not necessary to understandthe mechanism in order to use the present invention, and it is notintended that the present invention be so limited, one can contemplate anumber of mechanisms by which the delivery is enhanced by the presentinvention. In some instances cleavage of the labile polymer leads torelease and enhanced delivery of the therapeutic agent (biologicallyactive compound). Cleavage can also lead to enhanced membrane activityso that the pharmaceutical (biologically active compound) is moreeffectively delivered to the cell. This can occur in the environs of atumor or inflamed tissue or within an acidic sub-cellular compartment.Cleavage can also cause an osmotic shock to the endosomes and disruptsthe endosomes. If the polymer backbone is hydrophobic it may interactwith the membrane of the endosome. Either effect disrupts the endosomeand thereby assists in release of delivered compound.

In some embodiments of the present invention, membrane active agents arecomplexed with the delivery system such that they are inactive and notmembrane active within the complex but become active when released,following the chemical conversion of the labile group. The membraneactive agents may be used to assist in the disruption of the endosome orother cellular compartment. They can also be used to enable selectivedelivery or toxicity to tumors or tissues that are acidic. Many membraneactive agents such as the peptides melittin and pardaxin and variousviral proteins and peptides are effective in allowing a disruption ofcellular compartments such as endosomes to effect a release of itscontents into a cell. However, these agents are toxic to cells both invitro and in vivo due to the inherent nature of their membrane activity.To decrease the toxicity of these agents, the present invention providestechniques to complex or modify the agent in a way which blocks orinhibits the membrane activity of the agent but is reversible in natureso activity can be recovered when membrane activity is needed fortransport of biologically active compound. The activities of thesemembrane active agents can be controlled in a number of different ways.For example, a modification of the agent may be made that can be cleavedoff of the agent allowing the activity to return. The cleavage can occurduring a natural process, such as the pH drop seen in endosomes orcleaved in the cytoplasm of cells where amounts of reducing agentsbecome available. Cleavage of a blocking agent can occur by delivery ofa cleaving agent to the blocked complex at a time when it would be mostbeneficial. Another exemplary method of blocking membrane active agentsis to reversibly modify the agents' functional group with an activityblocking addition (defined as “Compounds or Chemical Moieties thatInhibit or Block the Membrane Activity of Another Compound or ChemicalMoiety”. When the blocking addition reaches an environment or an adjunctis added the reversible modification is reversed and the membrane activeagent will regain activity.

In some embodiments the biologically active compound is reversiblymodified, or complexed with, an interaction modifier such that theinteractions between the biologically active molecule and its environs,that is its interactions with itself and other molecules, is alteredwhen the interaction modifier is released. For example attachment ofsuch nonionic hydrophilic groups such as polyethylene glycol andpolysaccharides (e.g. starch) may decrease self-association andinteractions with other molecules such as serum compounds and cellularmembranes, which may be necessary for transport of the biologicallyactive molecule to the cell. However these molecules may inhibitcellular uptake and therefore, must be lost before cellular uptake canoccur. Likewise, cell targeting ligands aid in transport to a cell butmay not be necessary, and may inhibit, transport into a cell. In all ofthese cases, the reversible attachment of the interaction modifier,through a labile bond, would be beneficial.

The present invention provides for the transfer of polynucleotides, andother biologically active compounds into cells in culture (also known as“in vitro”). Compounds or kits for the transfection of cells in cultureis commonly sold as “transfection reagents” or “transfection kits”. Thepresent invention also provides for the transfer of polynucleotides, andbiologically active compounds into cells within tissues in situ and invivo, and delivered intravasculary (U.S. patent application Ser. No.08/571,536, abandoned), intrarterially, intravenous, orally,intraduodenaly, via the jejunum (or ileum or colon), rectally,transdermally, subcutaneously, intramuscularly, intraperitoneally,intraparenterally, via direct injections into tissues such as the liver,lung, heart, muscle, spleen, pancreas, brain (includingintraventricular), spinal cord, ganglion, lymph nodes, lymphatic system,adipose tissues, thryoid tissue, adrenal glands, kidneys, prostate,blood cells, bone marrow cells, cancer cells, tumors, eye retina, viathe bile duct, or via mucosal membranes such as in the mouth, nose,throat, vagina or rectum or into ducts of the salivary or other exocrineglands. Compounds for the transfection of cells in vivo in a wholeorganism can be sold as “in vivo transfection reagents” or “in vivotransfection kits” or as a pharmaceutical for gene therapy.

Polymers with pH-Labile Bonds

The present invention provides a wide variety of polymers with labilegroups that find use in the delivery systems of the present invention.The labile groups are selected such that they undergo a chemicaltransformation (e.g., cleavage) when present in physiologicalconditions. The chemical transformation may be initiated by the additionof a compound to the cell or may occur spontaneously when introducedinto intra-and/or extra-cellular environments (e.g., the lower pHconditions of an endosome or the extracellular space surroundingtumors). The conditions under which a labile group will undergotransformation can be controlled by altering the chemical constituentsof the molecule containing the labile group. For example, addition ofparticular chemical moieties (e.g., electron acceptors or donors) nearthe labile group can effect the particular conditions (e.g., pH) underwhich chemical transformation will occur.

In certain embodiments, the present invention provides compound deliverysystems composed of polymers (e.g., cationic polymers, anionic polymers,zwitterionic and nonionic polymers) that contain pH-labile groups. Thesystems are relatively chemically stable until they are introduced intoacidic conditions that render them unstable (labile). An aqueoussolution is acidic when the concentration of protons (H⁺) exceed theconcentration of hydroxide (OH⁻). Upon delivery to the desired location,the labile group undergoes an acid-catalyzed chemical transformationresulting in release of the delivered compound or a complex of thedelivered compound. The pH-labile bond may either be in the main-chainor in the side chain. If the pH-labile bond occurs in the main chain,then cleavage of the labile bond results in a decrease in polymerlength. If the pH-labile bond occurs in the side chain, then cleavage ofthe labile bond results in loss of side chain atoms from the polymer.

In some preferred embodiments of the present invention, nucleic acidsare delivered to cells by a polymer complex containing a labile group,or groups, that undergoes chemical transformation when exposed to thelow pH environment of an endosome. Such complexes provide improvednucleic acid delivery systems, as they provide for efficient deliveryand low toxicity.

Polymers Containing Several Membrane Active Compounds

The present invention specifies polymers containing more than twomembrane active compounds. In one embodiment, the membrane activecompounds are grafted onto a preformed polymer to form a comb-typepolymer, i.e. a polymer containing side chain groups. In anotherembodiment, the membrane active compounds are incorporated into thepolymer by chain or step polymerization processes. To aid incomplexation between DNA and membrane active compounds and/or to augmentthe membrane activity of membrane active agents, certain embodiments ofthe present invention have polymers composed of monomers that arethemselves membrane active. These polymers are formed by attaching amembrane active compound to a preformed polymer or by polymerization ofmembrane active monomers.

Membrane Active Compounds Containing Labile Bonds

The inclusion of labile bonds into membrane active compounds increasestheir versatility in a number of ways. It can reduce their toxicity byenabling their membrane activity to be expressed in specific tissuessuch as tumors and inflamed joints, specific sub-cellular locations suchas endosomes and lysosomes, or under specific conditions such as areducing environment. In one embodiment of the invention, the labilebonds are pH-sensitive in that the bonds break or are cleaved when pH oftheir microenvironment drops below physiologic pH of 7.4 or below pH of6.5 or below pH of 5.5. In another embodiment the labile bonds are verypH-sensitive. In yet another embodiment, the labile bonds are disulfidesthat are labile under physiologic conditions or that are cleaved by theaddition of an exogenous reducing agent. In other embodiments, thelabile bonds are acetals, ketals, enol ethers, enol esters, amides of2,3-disubstituted maleamic acid, imines, imminiums, enamines, silylethers, and silyl enol ethers.

The invention also includes compounds that are of the general structure:A-B-C wherein A is a membrane active compound, B is a labile linkage,and C is a compound that inhibits the membrane activity of compound A.Upon cleavage of B, membrane activity is restored to compound A. Thiscleavage occurs in certain tissue, organ, and sub-cellular locationsthat are controlled by the microenvironment of the location and also bythe addition of exogenous agents. In another embodiment, the inventionincludes compositions containing biologically active compounds andcompounds of the general structure: A-B-C wherein A is a membrane activecompound, B is a labile linkage, and C is a compound that inhibits themembrane activity of compound A. The biologically-active compoundsinclude pharmaceutical drugs, nucleic acids and genes. In yet anotherembodiment, these compounds that are of the general structure—A-B-Cwherein A is a membrane active compound, B is a labile linkage, and C isa compound that inhibits the membrane activity of compound A- .are usedto deliver biologically active compounds that include pharmaceuticaldrugs, nucleic acids and genes. In one specific embodiment, these A-B-Ccompounds are used to deliver nucleic acids and genes to muscle(skeletal, heart, respiratory, striated, and non-striated), liver(hepatocytes), spleen, immune cells, gastrointestinal cells, cells ofthe nervous system (neurons, glial, and microglial), skin cells (dermisand epidermis), joint and synovial cells, tumor cells, kidney, cells ofthe immune system (dendiritic, T cells, B cells, antigen-presentingcells, macrophages), exocrine cells (pancreas, salivary glands),prostate, adrenal gland, thyroid gland, eye structures (retinal cells),and respiratory cells (cells of the lung, nose, respiratory tract)

Mixtures of Membrane Active Compounds and Labile Compounds

In addition, the invention is a composition of matter that includes amembrane active compound and a labile compound. In one embodiment, thelabile compound inhibits the membrane activity of the membrane activecompound. Upon chemical modification of the labile compound, membraneactivity is restored to the membrane active compound. This chemicalmodification occurs in certain tissue, organ, and sub-cellular locationsthat are controlled by the microenvironment of the location and also bythe addition of exogenous agents. In one embodiment the chemicalmodification involves the cleavage of the polymer. In one embodiment,the membrane active compound and the inhibitory labile compound arepolyions and are of opposite charge. For example, the membrane activecompound is a polycation and the inhibitory labile compound is apolyanion.

In another embodiment, the invention includes compositions containingbiologically active compounds, a membrane active compound and a labilecompound. Upon chemical modification of the labile compound, membraneactivity is restored to the membrane active compound. This chemicalmodification occurs in certain tissue, organ, and sub-cellular locationsthat are controlled by the microenvironment of the location and also bythe addition of exogenous agents. In one embodiment the chemicalmodification involves the cleavage of the polymer. In one specificembodiment, these compositions containing biologically active compounds,a membrane active compound and a labile compound are used to delivernucleic acids and genes to muscle (skeletal, heart, respiratory,striated, and non-striated), liver (hepatocytes), spleen, immune cells,gastrointestinal cells, cells of the nervous system (neurons, glial, andmicroglial), skin cells (dermis and epidermis), joint and synovialcells, tumor cells, kidney, cells of the immune system (dendiritic, Tcells, B cells, antigen-presenting cells, macrophages), exocrine cells(pancreas, salivary glands), prostate, adrenal gland, thyroid gland, eyestructures (retinal cells), respiratory cells (cells of the lung, nose,respiratory tract), and endothelial cells.

Biologically Active Compounds Containing pH-Labile and/or Extremelyand/or Very pH-Labile Bonds

The invention specifies compounds of the following general structure:A-B-C wherein A is a biologically active compound such aspharmaceuticals, drugs, proteins, peptides, hormones, cytokines, enzymesand nucleic acids such as anti-sense, ribozyme, recombining nucleicacids, and expressed genes; B is a labile linkage that contains apH-labile bond such as acetals, ketals, enol ethers, enol esters, amidesof 2,3-disubstituted maleamic acids, imines, imminiums, enamines, silylethers, and silyl enol ethers; and C is a compound. In one embodiment Cis a compound that modifies the activity, function, delivery, transport,shelf-life, pharmacokinetics, blood circulation time in vivo, tissue andorgan targetting, and sub-cellular targeting of the biologically activecompound A. For example, C can be a hydrophilic compound such aspolyethylene glycol to increase the water solubility of relativelyhydrophobic drugs (e.g. amphotericin B) to improve their formulation anddelivery properties. In other embodiments, B is a labile linkage thatcontains pH-labile bond such as acetals, ketals, enol ethers, enolesters, amides, imines, imminiums, enamines, silyl ethers, and silylenol ethers.

The invention also specifies that the labile linkage B is attached toreactive functional groups on the biologically active compound A. In yetanother embodiment, reactive functional groups are attached to nucleicacids via alkylation. Specifically, nitrogen and sulfur mustards may beused for modify nucleic acids with reactive functional groups.

pH-Labile Amphipathic Compounds

In one specification of the invention, the pH-labile and very pH-labilelinkages and bonds are used within amphipathic compounds and detergents.The pH-labile amphipathic compounds can be incorporated into liposomesfor delivery of biologically active compounds and nucleic acids tocells. The detergents can be used for cleaning purposes and formodifying the solubility of biologically active compounds such asproteins. The detergents can be in the form of micelles or reversemicelles. Often detergents are used to extract biologically activecompounds from natural mixtures. After the extraction procedure iscompleted, a labile detergent would aid in the separation of thedetergent and the biologically active compound. If the detergent islabile under conditions that do not harm the biologically activecompound (e.g. destroying or denaturing a protein), then removal of thedetergent would be much easier that currently-used methods.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

Biologically Active Compound

A biologically active compound is a compound having the potential toreact with biological components. More particularly, biologically activecompounds utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. In this specification, the cellular production of, orinhibition of a material, such as a protein, caused by a human assistinga molecule to an in vivo cell is an example of a delivered biologicallyactive compound. Pharmaceuticals, proteins, peptides, polypeptides,enzyme inhibitors, hormones, cytokines, antigens, viruses,oligonucleotides, enzymes and nucleic acids are examples of biologicallyactive compounds.

Peptide and polypeptide refer to a series of amino acid residues, morethan two, connected to one another by amide bonds between the beta oralpha-amino group and carboxyl group of contiguous amino acid residues.The amino acids may be naturally occurring or synthetic. Polypeptideincludes proteins and peptides, modified proteins and peptides, andnon-natural proteins and peptides. Enzymes are proteins evolved by thecells of living organisms for the specific function of catalyzingchemical reactions. A chemical reaction is defined as the formation orcleavage of covalent or ionic bonds. Bioactive compounds may be usedinterchangeably with biologically active compound for purposes of thisapplication.

Delivery of Biologically Active Compound

The delivery of a biologically active compound is commonly known as“drug delivery”. “Delivered” means that the biologically active compoundbecomes associated with the cell or organism. The compound can be in thecirculatory system, intravessel, extracellular, on the membrane of thecell or inside the cytoplasm, nucleus, or other organelle of the cell.

Parenteral routes of administration include intravascular (intravenous,intraarterial), intramuscular, intraparenchymal, intradermal, subdermal,subcutaneous, intratumor, intraperitoneal, intrathecal, subdural,epidural, and intralymphatic injections that use a syringe and a needleor catheter. An intravascular route of administration enables a polymeror polynucleotide to be delivered to cells more evenly distributed andmore efficiently expressed than direct injections. Intravascular hereinmeans within a tubular structure called a vessel that is connected to atissue or organ within the body. Within the cavity of the tubularstructure, a bodily fluid flows to or from the body part. Examples ofbodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid,or bile. Examples of vessels include arteries, arterioles, capillaries,venules, sinusoids, veins, lymphatics, and bile ducts. The intravascularroute includes delivery through the blood vessels such as an artery or avein. An administration route involving the mucosal membranes is meantto include nasal, bronchial, inhalation into the lungs, or via the eyes.Other routes of administration include intraparenchymal into tissuessuch as muscle (intramuscular), liver, brain, and kidney. Transdermalroutes of administration have been effected by patches andionotophoresis. Other epithelial routes include oral, nasal,respiratory, and vaginal routes of administration.

Delivery System

Delivery system is the means by which a biologically active compoundbecomes delivered. That is all compounds, including the biologicallyactive compound itself, that are required for delivery and allprocedures required for delivery including the form (such volume andphase (solid, liquid, or gas)) and method of administration (such as butnot limited to oral or subcutaneous methods of delivery).

Nucleic Acid

The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and naturalanalogs, and synthetic derivatives of purines and pyrimidines, whichinclude, but are not limited to, modifications which place new reactivegroups such as, but not limited to, amines, alcohols, thiols,carboxylates, and alkylhalides. Nucleotides are the monomeric units ofnucleic acid polymers. A “polynucleotide” is distinguished here from an“oligonucleotide” by containing more than 80 monomeric units;oligonucleotides contain from 2 to 80 nucleotides. The term nuclei acidincludes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

DNA may be in the form of anti-sense, plasmid DNA, parts of a plasmidDNA, product of a polymerase chain reaction (PCR), vectors (P1, PAC,BAC, YAC, artificial chromosomes), expression cassettes, chimericsequences, chromosomal DNA, or derivatives of these groups. RNA may bein the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (smallnuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-senseRNA, ribozymes, chimeric sequences, or derivatives of these groups.

“Anti-sense” is a polynucleotide that interferes with the function ofDNA and/or RNA. This may result in suppression of expression. Naturalnucleic acids have a phosphate backbone, artificial nucleic acids maycontain other types of backbones and bases. These include PNAs (peptidenucleic acids), phosphothionates, and other variants of the phosphatebackbone of native nucleic acids. In addition, DNA and RNA may besingle, double, triple, or quadruple stranded.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques. “Expression cassette” refers to anatural or recombinantly produced polynucleotide molecule that iscapable of expressing protein(s). A DNA expression cassette typicallyincludes a promoter (allowing transcription initiation), and a sequenceencoding one or more proteins. Optionally, the expression cassette mayinclude transcriptional enhancers, non-coding sequences, splicingsignals, transcription termination signals, and polyadenylation signals.An RNA expression cassette typically includes a translation initiationcodon (allowing translation initiation), and a sequence encoding one ormore proteins. Optionally, the expression cassette may includetranslation termination signals, a polyadenosine sequence, internalribosome entry sites (IRES), and non-coding sequences.

A nucleic acid can be used to modify the genomic or extrachromosomal DNAsequences. This can be achieved by delivering a nucleic acid that isexpressed. Alternatively, the nucleic acid can effect a change in theDNA or RNA sequence of the target cell. This can be achieved byhomologous recombination, gene conversion, or other yet to be describedmechanisms.

Gene

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., -myosin heavy chain). The polypeptide can be encodedby a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ non-translated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequencewhich encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form.

Gene Expression

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decreases production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

Delivery of Nucleic Acids

The process of delivering a polynucleotide to a cell has been commonlytermed “transfection” or the process of “transfecting” and also it hasbeen termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The delivery of nucleic acidcan lead to modification of the DNA sequence of the target cell.

The polynucleotides or genetic material being delivered are generallymixed with transfection reagents prior to delivery. The term“transfection” as used herein refers to the introduction of foreign DNAinto eukaryotic cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas irreversibly integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA. The term “naked polynucleotides”indicates that the polynucleotides are not associated with atransfection reagent or other delivery vehicle that is required for thepolynucleotide to be delivered to a cell.

A “transfection reagent” or “delivery vehicle” is a compound orcompounds that bind(s) to or complex(es) with oligonucleotides,polynucleotides, or other desired compounds and mediates their entryinto cells. Examples of transfection reagents include, but are notlimited to, cationic liposomes and lipids, polyamines, calcium phosphateprecipitates, histone proteins, polyethylenimine, and polylysinecomplexes (polyethylenimine and polylysine are both toxic). Typically,when used for the delivery of nucleic acids, the transfection reagenthas a net positive charge that binds to the polynucleotide's negativecharge. For example, cationic liposomes or polylysine complexes have netpositive charges that enable them to bind to DNA or RNA.

Enzyme

Enzyme is a protein that acts as a catalyst. That is a protein thatincreases the rate of a chemical reaction without itself undergoing anypermanent chemical change. The chemical reactions that are catalyzed byan enzyme are termed enzymatic reactions and chemical reactions that arenot are termed nonenzymatic reactions.

Half-Life

The half-life of a chemical reaction is the time required for one halfof a given material to undergo a chemical reaction.

Complex

Two molecules are combined, to form a complex through a process calledcomplexation or complex formation, if the are in contact with oneanother through noncovalent interactions such as electrostaticinteractions, hydrogen bonding interactions, and hydrophobicinteractions.

Modification

A molecule is modified, to form a modification through a process calledmodification, by a second molecule if the two become bonded through acovalent bond. That is, the two molecules form a covalent bond betweenan atom form one molecule and an atom from the second molecule resultingin the formation of a new single molecule. A chemical covalent bond isan interaction, bond, between two atoms in which there is a sharing ofelectron density.

Osmosis

Osmosis is the passage of a solvent through a semipermeable membrane, amembrane through which solvent can pass but not all solutes, separatingtwo solutions of different concentrations. There is a tendency for theseparated solutions to become the same concentration as the solventpasses from low concentration to high concentration. Osmosis will stopwhen the two solutions become equal in concentration or when pressure isapplied to the solution containing higher concentration. When the higherconcentrated solution is in a closed system, that is when system is ofconstant volume, there is a build up of pressure as the solvent passesfrom low to high concentration. This build up of pressure is calledosmotic pressure.

Salt

A salt is any compound containing ionic bonds, that is bonds in whichone or more electrons are transferred completely from one atom toanother.

Interpolyelectrolyte Complexes

An interpolyelectrolyte complex is a noncovalent interaction betweenpolyelectrolytes of opposite charge.

Charge, Polarity, and Sign

The charge, polarity, or sign of a compound refers to whether or not acompound has lost one or more electrons (positive charge, polarity, orsign) or gained one or more electrons (negative charge, polarity, orsign).

Cell Targeting Signals

Cell targeting signal (or abbreviated as the Signal) is defined in thisspecification as a molecule that modifies a biologically activecompounds such as drug or nucleic acid and can direct it to a celllocation (such as tissue) or location in a cell (such as the nucleus)either in culture or in a whole organism. By modifying the cellular ortissue location of the foreign gene, the function of the biologicallyactive compound can be enhanced.

The cell targeting signal can be a protein, peptide, lipid, steroid,sugar, carbohydrate, (non-expressing) polynucleic acid or syntheticcompound. The cell targeting signal enhances cellular binding toreceptors, cytoplasmic transport to the nucleus and nuclear entry orrelease from endosomes or other intracellular vesicles.

Nuclear localizing signals enhance the targeting of the pharmaceuticalinto proximity of the nucleus and/or its entry into the nucleus. Suchnuclear transport signals can be a protein or a peptide such as the SV40large T ag NLS or the nucleoplasmin NLS. These nuclear localizingsignals interact with a variety of nuclear transport factors such as theNLS receptor (karyopherin alpha) which then interacts with karyopherinbeta. The nuclear transport proteins themselves could also function asNLS's since they are targeted to the nuclear pore and nucleus. Forexample, karyopherin beta itself could target the DNA to the nuclearpore complex. Several peptides have been derived from the SV40 Tantigen. These include a short NLS (H-SEQ ID NO: 2-OH,) or long NLS's(H-SEQ ID NO: 3-OH,; and H-SEQ ID NO: 4-OH,). Other NLS peptides havebeen derived from M9 protein (SEQ ID NO: 5), E1A (H-SEQ ID NO: 6-OH,),nucleoplasmin (H-SEQ ID NO: 7-OH,), and c-myc (H-SEQ ID NO: 8-OH,).

Signals that enhance release from intracellular compartments (releasingsignals) can cause DNA release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Releasingsignals include chemicals such as chloroquine, bafilomycin or BrefeldinA1 and the ER-retaining signal (SEQ ID NO: 9), viral components such asinfluenza virus hemagglutinin subunit HA-2 peptides and other types ofamphipathic peptides.

Cellular receptor signals are any signal that enhances the associationof the biologically active compound with a cell. This can beaccomplished by either increasing the binding of the compound to thecell surface and/or its association with an intracellular compartment,for example: ligands that enhance endocytosis by enhancing binding thecell surface. This includes agents that target to the asialoglycoproteinreceptor by using asiologlycoproteins or galactose residues. Otherproteins such as insulin, EGF, or transferrin can be used for targeting.Peptides that include the RGD sequence can be used to target many cells.Chemical groups that react with thiol, sulfhydryl, or disulfide groupson cells can also be used to target many types of cells. Folate andother vitamins can also be used for targeting. Other targeting groupsinclude molecules that interact with membranes such as lipids, fattyacids, cholesterol, dansyl compounds, and amphotericin derivatives. Inaddition viral proteins could be used to bind cells.

Interaction Modifiers

An interaction modifier changes the way that a molecule interacts withitself or other molecules, relative to molecule containing nointeraction modifier. The result of this modification is thatself-interactions or interactions with other molecules are eitherincreased or decreased. For example cell targeting signals areinteraction modifiers with change the interaction between a molecule anda cell or cellular component. Polyethylene glycol is an interactionmodifier that decreases interactions between molecules and themselvesand with other molecules.

Reporter or Marker Molecules

Reporter or marker molecules are compounds that can be easily detected.Typically they are fluorescent compounds such as fluorescein, rhodamine,Texas red, cy 5, cy 3 or dansyl compounds. They can be molecules thatcan be detected by infrared, ultraviolet or visible spectroscopy or byantibody interactions or by electron spin resonance. Biotin is anotherreporter molecule that can be detected by labeled avidin. Biotin couldalso be used to attach targeting groups.

Linkages

An attachment that provides a covalent bond or spacer between two othergroups (chemical moieties). The linkage may be electronically neutral,or may bear a positive or negative charge. The chemical moieties can behydrophilic or hydrophobic. Preferred spacer groups include, but are notlimited to C1–C12 alkyl, C1–C12 alkenyl, C1–C12 alkynyl, C6–C18 aralkyl,C6–C18 aralkenyl, C6–C18 aralkynyl, ester, ether, ketone, alcohol,polyol, amide, amine, polyglycol, polyether, polyamine, thiol, thioether, thioester, phosphorous containing, and heterocyclic.

Bifunctional

Bifunctional molecules, commonly referred to as crosslinkers, are usedto connect two molecules together, i.e. form a linkage between twomolecules. Bifunctional molecules can contain homo orheterobifunctionality.

Crosslinking

Crosslinking refers to the chemical attachment of two or more moleculeswith a bifunctional reagent. A bifunctional reagent is a molecule withtwo reactive ends. The reactive ends can be identical as in ahomobifunctional molecule, or different as in a heterobifunctionalmolecule.

Labile Bond

A labile bond is a covalent bond that is capable of being selectivelybroken. That is, the labile bond may be broken in the presence of othercovalent bonds without the breakage of other covalent bonds. Forexample, a disulfide bond is capable of being broken in the presence ofthiols without cleavage of any other bonds, such as carbon-carbon,carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also bepresent in the molecule.

Labile Linkage

A labile linkage is a chemical compound that contains a labile bond andprovides a link or spacer between two other groups. The groups that arelinked may be chosen from compounds such as biologically activecompounds, membrane active compounds, compounds that inhibit membraneactivity, functional reactive groups, monomers, and cell targetingsignals. The spacer group may contain chemical moieties chosen from agroup that includes alkanes, alkenes, esters, ethers, glycerol, amide,saccharides, polysaccharides, and heteroatoms such as oxygen, sulfur, ornitrogen. The spacer may be electronically neutral, may bear a positiveor negative charge, or may bear both positive and negative charges withan overall charge of neutral, positive or negative.

pH-Labile Linkages and Bonds

pH-labile refers to the selective breakage of a covalent bond underacidic conditions (pH<7). That is, the pH-labile bond may be brokenunder acidic conditions in the presence of other covalent bonds withouttheir breakage. The term pH-labile includes both linkages and bonds thatare pH-labile, very pH-labile, and extremely pH-labile.

Very pH-Labile Linkages and Bonds

A subset of pH-labile bonds is very pH-labile. For the purposes of thepresent invention, a bond is considered very pH-labile if the half-lifefor cleavage at pH 5 is less than 45 minutes.

Extremely pH-Labile Linkages and Bonds

A subset of pH-labile bonds is extremely pH-labile. For the purposes ofthe present invention, a bond is considered extremely pH-labile if thehalf-life for cleavage at pH 5 is less than 15 minutes.

Amphiphilic and Amphipathic Compounds

Amphipathic, or amphiphilic, compounds have both hydrophilic(water-soluble) and hydrophobic (water-insoluble) parts. Hydrophilicgroups indicate in qualitative terms that the chemical moiety iswater-preferring. Typically, such chemical groups are water soluble, andare hydrogen bond donors or acceptors with water. Examples ofhydrophilic groups include compounds with the following chemicalmoieties; carbohydrates, polyoxyethylene, peptides, oligonucleotides andgroups containing amines, amides, alkoxy amides, carboxylic acids,sulfurs, or hydroxyls. Hydrophobic groups indicate in qualitative termsthat the chemical moiety is water-avoiding. Typically, such chemicalgroups are not water soluble, and tend not to hydrogen bonds.Hydrocarbons are hydrophobic groups.

Detergent

Detergents or surfactants are water-soluble molecules containing ahydrophobic portion (tail) and a hydrophilic portion (head), which uponaddition to water decrease water's surface tension. The hydrophobicportion can be alkyl, alkenyl, alkynyl or aromatic. The hydrophilicportion can be charged with either net positive (cationic detergents),negative (anionic detergents), uncharged (nonionic detergents), orcharge neutral (zwitterionic detergent). Examples of anionic detergentsare sodium dodecyl sulfate, glycolic acid ethoxylate(4 units)4-tert-butylphenylether, palmitic acid, and oleic acid. Examples ofcationic detergents are cetyltrimethylammonium bromide and oleylamine.Examples of nonionic detergents include, laurylmaltoside, Triton X-100,and Tween. Examples of zwitterionic detergents include3-[(3-cholamidopropyl)dimthylammonio]1-propane-sulfonate (CHAPS), andN-tetradecyl-N,N-dimethyl-3-ammoniu-1-propanesulfonate.

Surface Tension

The surface tension of a liquid is the force acting over the surface ofthe liquid per unit length of surface that is perpendicular to the forcethat is acting of the surface. Surface charge has the units force perlength, e.g. Newtons/meter.

Membrane Active Compound

Membrane active agents or compounds are compounds (typically a polymer,peptide or protein) that are able alter the membrane structure. Thischange in structure can be shown by the compound inducing one or more ofthe following effects upon a membrane: an alteration that allows smallmolecule permeability, pore formation in the membrane, a fusion and/orfission of membranes, an alteration that allows large moleculepermeability, or a dissolving of the membrane. This alteration can befunctionally defined by the compound's activity in at least one thefollowing assays: red blood cell lysis (hemolysis), liposome leakage,liposome fusion, cell fusion, cell lysis and endosomal release. Anexample of a membrane active agent in our examples is the peptidemelittin, whose membrane activity is demonstrated by its ability torelease heme from red blood cells (hemolysis). In addition,dimethylmaleamic-modified melittin(DM-Mel) reverts to melittin in theacidic environment of the endosome causes endosomal release as seen bythe diffuse staining of fluorescein-labeled dextran in our endosomalrelease assay.

More specifically membrane active compounds allow for the transport ofmolecules with molecular weight greater than 50 atomic mass units tocross a membrane. This transport may be accomplished by either the totalloss of membrane structure, the formation of holes (or pores) in themembrane structure, or the assisted transport of compound through themembrane. In addition, transport between liposomes, or cell membranes,may be accomplished by the fusion of the two membranes and thereby themixing of the contents of the two membranes.

Membrane Active Peptides.

Membrane active peptides are peptides that have membrane activity. Thereare many naturally occurring membrane active peptides such as cecropin(insects), magainin, CPF 1, PGLa, Bombinin BLP-1 (all three fromamphibians), melittin (bees), seminalplasmin (bovine), indolicidin,bactenecin (both from bovine neutrophils), tachyplesin 1 (crabs),protegrin (porcine leukocytes), and defensins (from human, rabbit,bovine, fungi, and plants). Gramicidin A and gramicidin S (bacillusbrevis), the lantibiotics such as nisin (lactococcus lactis),androctonin (scorpion), cardiotoxin I (cobra), caerin (frog litoriasplendida), dermaseptin (frog). Viral peptides have also been shown tohave membrane activity, examples include hemagglutinin subunit HA-2(influenza virus), E1 (Semliki forest virus), F1 (Sendai and measlesviruses), gp41 (HIV), gp32 (SIV), and vp1 (Rhino, polio, and coxsackieviruses). In addition synthetic peptides have also been shown to havemembrane activity. Synthetic peptides that are rich in leucines andlysines (KL or KL_(n) motif) have been shown to have membrane activity.In particular, the peptide H₂N-SEQ ID NO: 10-CO₂, termed KL₃, ismembrane active.

Compounds or Chemical Groups (Moieties) that Inhibit or Block theMembrane Activity of another Compound or Chemical Moiety

An interaction with a membrane active agent by modification orcomplexation (including covalent, ionic, hydrogen bonding, coordination,and van der Waals bonds) with another compound that causes a reduction,or cessation of the said agents membrane activity. Examples include thecovalent modification of a membrane-active peptide by the covalentattachment of an inhibitory chemical group (moiety) to the membraneactive peptide. Another example includes the interpolyelectrolytecomplexation of a membrane active polyanion and inhibitory polycation.

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

The main chain of a polymer is composed of the atoms whose bonds arerequired for propagation of polymer length. For example inpoly-L-lysine, the carbonyl carbon, α-carbon, and α-amine groups arerequired for the length of the polymer and are therefore main chainatoms. The side chain of a polymer is composed of the atoms whose bondsare not required for propagation of polymer length. For example inpoly-L-lysine, the β, γ, δ, and ε-carbons, and ε-nitrogen are notrequired for the propagation of the polymer and are therefore side chainatoms.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer. “Most step-reactionpolymerizations are condensation processes and most chain-reactionpolymerizations are addition processes” (M. P. Stevens PolymerChemistry: An Introduction New York Oxford University Press 1990).Template polymerization can be used to form polymers from daughterpolymers. Step Polymerization:

In step polymerization, the polymerization occurs in a stepwise fashion.Polymer growth occurs by reaction between monomers, oligomers andpolymers. No initiator is needed since there is the same reactionthroughout and there is no termination step so that the end groups arestill reactive. The polymerization rate decreases as the functionalgroups are consumed. Typically, step polymerization is done either oftwo different ways. One way, the monomer has both reactive functionalgroups (A and B) in the same molecule so thatA-B yields-[A-B]-Or the other approach is to have two difunctional monomers.A—A+B—B yields -[A—A−B—B]-Generally, these reactions can involve acylation or alkylation.Acylation is defined as the introduction of an acyl group (—COR) onto amolecule. Alkylation is defined as the introduction of an alkyl grouponto a molecule.

If functional group A is an amine then B can be (but not restricted to)an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide,sulfonyl chloride, aldehyde (including formaldehyde and glutaraldehyde),ketone, epoxide, carbonate, imidoester, carboxylate, or alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydrides or acid halides,p-nitrophenyl esters, o-nitrophenyl pentachlorophenyl esters, orpentafluorophenyl esters. In other terms when function A is an aminethen function B can be acylating or alkylating agent or amination.

If functional group A is a thiol, sulfhydryl, then function B can be(but not restricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group A is carboxylate then function B can be (but notrestricted to) a diazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylaminopyridine, N-hydroxysuccinimide or alcohol usingcarbodiimide and dimethylaminopyridine.

If functional group A is a hydroxyl then function B can be (but notrestricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used.

If functional group A is an aldehyde or ketone then function B can be(but not restricted to) an hydrazine, hydrazide derivative, amine (toform a imine or iminium that may or may not be reduced by reducingagents such as NaCNBH₃) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one difunctional monomer so thatA—A plus another agent yields-[A—A]-.If function A is a thiol, sulfhydryl, group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I₂) or NaIO₄ (sodiumperiodate), or oxygen (O₂). Function A can also be an amine that isconverted to a thiol, sulfhydryl, group by reaction with 2-Iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid {TNB} derivatives) can also be used tocatalyze disulfide bond formation.

Functional group A or B in any of the above examples could also be aphotoreactive group such as aryl azides, halogenated aryl azides, diazo,benzophenones, alkynes or diazirine derivatives.

Reactions of the amine, hydroxyl, thiol, sulfhydryl, carboxylate groupsyield chemical bonds that are described as amide, amidine, disulfide,ethers, esters, enamine, urea, isothiourea, isourea, sulfonamide,carbamate, carbon-nitrogen double bond (imine), alkylamine bond(secondary amine), carbon-nitrogen single bonds in which the carboncontains a hydroxyl group, thio-ether, diol, hydrazone, diazo, orsulfone.

Chain Polymerization: In chain-reaction polymerization growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.

Monomers containing vinyl, acrylate, methacrylate, acrylamide,methacrylamide groups can undergo chain reaction, which can be radical,anionic, or cationic. Chain polymerization can also be accomplished bycycle or ring opening polymerization. Several different types of freeradical initiatiors could be used that include peroxides, hydroxyperoxides, and azo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP). A compound is a material made up of two or moreelements.

Types of Monomers: A wide variety of monomers can be used in thepolymerization processes. These include positive charged organicmonomers such as amines, imidine, guanidine, imine, hydroxylamine,hydrazine, heterocycles (like imidazole, pyridine, morpholine,pyrimidine, or pyrene. The amines could be pH-sensitive in that thepK_(a) of the amine is within the physiologic range of 4 to 8. Specificamines include spermine, spermidine,N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

Monomers can also be hydrophobic, hydrophilic or amphipathic. Monomerscan also be intercalating agents such as acridine, thiazole organge, orethidium bromide.

Other Components of the Monomers and Polymers: The polymers have othergroups that increase their utility. These groups can be incorporatedinto monomers prior to polymer formation or attached to the polymerafter its formation. These groups include: Targeting Groups—such groupsare used for targeting the polymer-nucleic acid complexes to specificcells or tissues. Examples of such targeting agents include agents thattarget to the asialoglycoprotein receptor by using asiologlycoproteinsor galactose residues. Other proteins such as insulin, EGF, ortransferrin can be used for targeting. Protein refers to a molecule madeup of 2 or more amino acid residues connected one to another as in apolypeptide. The amino acids may be naturally occurring or synthetic.Peptides that include the RGD sequence can be used to target many cells.Chemical groups that react with thiol, sulfhydryl, or disulfide groupson cells can also be used to target many types of cells. Folate andother vitamins can also be used for targeting. Other targeting groupsinclude molecules that interact with membranes such as fatty acids,cholesterol, dansyl compounds, and amphotericin derivatives.

After interaction of the supramolecular complexes with the cell, othertargeting groups can be used to increase the delivery of the drug ornucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus.

A variety of ligands have been used to target drugs and genes to cellsand to specific cellular receptors. The ligand may seek a target withinthe cell membrane, on the cell membrane or near a cell. Binding ofligands to receptors typically initiates endocytosis. Ligands could alsobe used for DNA delivery that bind to receptors that are notendocytosed. For example peptides containing RGD peptide sequence thatbind integrin receptor could be used. In addition viral proteins couldbe used to bind the complex to cells. Lipids and steroids could be usedto directly insert a complex into cellular membranes.

The polymers can also contain cleavable groups within themselves. Whenattached to the targeting group, cleavage leads to reduce interactionbetween the complex and the receptor for the targeting group. Cleavablegroups include but are not restricted to disulfide bonds, diols, diazobonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enolesters, enamines and imines.

Polyelectrolyte

A polyelectrolyte, or polyion, is a polymer possessing charge, i.e. thepolymer contains a group (or groups) that has either gained or lost oneor more electrons. A polycation is a polyelectrolyte possessing netpositive charge, for example poly-L-lysine hydrobromide. The polycationcan contain monomer units that are charge positive, charge neutral, orcharge negative, however, the net charge of the polymer must bepositive. A polycation also can mean a non-polymeric molecule thatcontains two or more positive charges. A polyanion is a polyelectrolytecontaining a net negative charge. The polyanion can contain monomerunits that are charge negative, charge neutral, or charge positive,however, the net charge on the polymer must be negative. A polyanion canalso mean a non-polymeric molecule that contains two or more negativecharges. The term polyelectrolyte includes polycation, polyanion,zwitterionic polymers, and neutral polymers. The term zwitterionicrefers to the product (salt) of the reaction between an acidic group anda basic group that are part of the same molecule.

Chelator

A chelator is a polydentate ligand, a molecule that can occupy more thanone site in the coordination sphere of an ion, particularly a metal ion,primary amine, or single proton. Examples of chelators include crownethers, cryptates, and non-cyclic polydentate molecules. A crown etheris a cyclic polyether containing (—X—(CR1–2)n)m units, where n=1–3 andm=3–8. The X and CR1–2 moieties can be substituted, or at a differentoxidation states. X can be oxygen, nitrogen, or sulfur, carbon,phosphorous or any combination thereof. R can be H, C, O, S, N, P. Asubset of crown ethers described as a cryptate contain a second(—X—(CR1–2)n)z strand where z=3–8. The beginning X atom of the strand isan X atom in the (—X—(CR1–2)n)m unit, and the terminal CH2 of the newstrand is bonded to a second X atom in the (—X—(CR1–2)n)m unit.Non-cyclic polydentate molecules containing (—X—(CR1–2)n)m unit(s),where n=1–4 and m=1–8. The X and CR1–2 moieties can be substituted, orat a different oxidation states. X can be oxygen, nitrogen, or sulfur,carbon, phosphorous or any combination thereof.

Polychelator

A polychelator is a polymer associated with a plurality of chelators byan ionic or covalent bond and can include a spacer. The polymer can becationic, anionic, zwitterionic, neutral, or contain any combination ofcationic, anionic, zwitterionic, or neutral groups with a net chargebeing cationic, anionic or neutral, and may contain steric stabilizers,peptides, proteins, signals, or amphipathic compound for the formationof micellar, reverse micellar, or unilamellar structures. Preferably theamphipathic compound can have a hydrophilic segment that is cationic,anionic, or zwitterionic, and can contain polymerizable groups, and ahydrophobic segment that can contain a polymerizable group.

Steric Stabilizer

A steric stabilizer is a long chain hydrophilic group that preventsaggregation of final polymer by sterically hindering particle toparticle electrostatic interactions. Examples include: alkyl groups, PEGchains, polysaccharides, hydrogen molecules, alkyl amines. Electrostaticinteractions are the non-covalent association of two or more substancesdue to attractive forces between positive and negative charges.

Buffers

Buffers are made from a weak acid or weak base and their salts. Buffersolutions resist changes in pH when additional acid or base is added tothe solution.

Biological, Chemical, or Biochemical Reactions

Biological, chemical, or biochemical reactions involve the formation orcleavage of ionic and/or covalent bonds.

Reactive

A compound is reactive if it is capable of forming either an ionic or acovalent bond with another compound. The portions of reactive compoundsthat are capable of forming covalent bonds are referred to as reactivefunctional groups.

Lipids

Lipids are compounds that are insoluble in water but soluble in organicsolvent which have the general structure composed of two distincthydrophobic sections, that is two separate sections of uninterruptedcarbon-carbon bonds. The two hydrophobic sections are connected througha linkage that contains at least one heteroatom, that is an atom that isnot carbon (e.g. nitrogen, oxygen, silicon, and sulfur). Examplesinclude esters and amides of fatty acids and include the glycerides(1,2-dioleoylglycerol (DOG)), glycolipids, phospholipids(dioleoylphosphatidylethanolamine (DOPE)).

Hydrocarbon

Hydrocarbon means containing carbon and hydrogen atoms; andhalohydrocarbon means containing carbon, halogen (F, Cl, Br, I), andhydrogen atoms.

Alkyl, Alkene, Alkyne, Aryl

Alkyl means any sp³-hybridized carbon-containing group; alkenyl meanscontaining two or more sp² hybridized carbon atoms; aklkynyl meanscontaining two or more sp hybridized carbon atoms; aralkyl meanscontaining one or more aromatic ring(s) in addition containing sp³hybridized carbon atoms; aralkenyl means containing one or more aromaticring(s) in addition to containing two or more sp² hybridized carbonatoms; aralkynyl means containing one or more aromatic ring(s) inaddition to containing two or more sp hybridized carbon atoms; steroidincludes natural and unnatural steroids and steroid derivatives.

Steroid

A steroid derivative means a sterol, a sterol in which the hydroxylmoiety has been modified (for example, acylated), or a steroid hormone,or an analog thereof. The modification can include spacer groups,linkers, or reactive groups.

Carbohydrate

Carbohydrates include natural and unnatural sugars (for exampleglucose), and sugar derivatives (a sugar derivative means a system inwhich one or more of the hydroxyl groups on the sugar moiety has beenmodified (for example, but not limited to, acylated), or a system inwhich one or more of the hydroxyl groups is not present).

Polyoxyethylene

Polyoxyethylene means a polymer having ethylene oxide units(—(CH₂CH₂O)_(n)—, where n=2–3000).

Compound

A compound is a material made up of two or more elements.

Electron Withdrawing and Donating Groups

Electron withdrawing group is any chemical group or atom composed ofelectronegative atom(s), that is atoms that tend to attract electrons.Electron donating group is any chemical group or atom composed ofelectropositive atom(s), that is atoms that tend to attract electrons.

Resonance Stabilization

Resonance stabilization is the ability to distribute charge on multipleatoms through pi bonds. The inductive effective, in a molecule, is ashift of electron density due to the polarization of a bond by a nearbyelectronegative or electropositive atom.

Sterics

Steric hindrance, or sterics, is the prevention or retardation of achemical reaction because of neighboring groups on the same molecule.

Activated Carboxylate

An activated carboxylate is a carboxylic acid derivative that reactswith nucleophiles to form a new covalent bond. Nucleophiles includenitrogen, oxygen and sulfur-containing compounds to produce ureas,amides, carbonates, carbamates, esters, and thioesters. The carboxylicacid may be activated by various agents including carbodiimides,carbonates, phosphoniums, and uroniums to produce activated carboxylatesacyl ureas, acylphosphonates, acid anhydrides, and carbonates.Activation of carboxylic acid may be used in conjunction with hydroxyand amine-containing compounds to produce activated carboxylatesN-hydroxysuccinimide esters, hydroxybenzotriazole esters,N-hydroxy-5-norbornene-endo-2,3-dicarboximide esters, p-nitrophenylesters, pentafluorophenyl esters, 4-dimethylaminopyridinium amides, andacyl imidazoles.

Nucleophile

A nucleophile is a species possessing one or more electron-rich sites,such as an unshared pair of electrons, the negative end of a polar bond,or pi electrons.

Cleavage and Bond Breakage

Cleavage, or bond breakage is the loss of a covalent bond between twoatoms. Cleavable means that a bond is capable of being cleaved.

Substituted Group or Substitution

A substituted group or a substitution refers to chemical group that isplaced onto a parent system instead of a hydrogen atom. For the compoundmethylbenzene (toluene), the methyl group is a substituted group, orsubstitution on the parent system benzene. The methyl groups on2,3-dimethylmaleic anhydride are substituted groups, or substitutions onthe parent compound (or system) maleic anhydride.

Primary and Secondary Amine

A primary amine is a nitrogen-containing compound that is derived bymonosubstitution of ammonia (NH₃) by a carbon-containing group. Aprimary amine is a nitrogen-containing compound that is derived bydisubstitution of ammonia (NH₃) by a carbon-containing group.

Preferred Embodiments

The following description provides exemplary embodiments of the systems,compositions, and methods of the present invention. These embodimentsinclude a variety of systems that have been demonstrated as effectivedelivery systems both in vitro and in vivo. The invention is not limitedto these particular embodiments. The following topics are discussed inturn: I) Labile, pH-labile, Very pH-labile Bonds, and ExtremelypH-Labile Bonds II) Polymers with pH-Labile Bonds, III) PolymersContaining Several Membrane Active Compounds, IV) Membrane ActiveCompounds Containing Labile Bonds, V) Mixtures of Membrane ActiveCompounds and Labile Compounds, VI) Biologically active compoundsContaining Very pH-Labile Bonds, and VII) pH-Labile AmphipathicCompounds

I. Labile, pH-Labile Bonds, Very pH-Labile, and Extremely pH-LabileBonds

A) Labile Bonds

In one embodiment, disulfide bonds are used in a variety of molecules,and polymers that include peptides, lipids, liposomes.

B) pH-Labile

In one embodiment, ketals that are labile in acidic environments (pHless than 7, greater than 4) to form a diol and a ketone are used in avariety of molecules and polymers that include peptides, lipids, andliposomes.

In one embodiment, acetals that are labile in acidic environments (pHless than 7, greater than 4) to form a diol and an aldehyde are used ina variety of molecules and polymers that include peptides, lipids, andliposomes.

In one embodiment, imines or iminiums that are labile in acidicenvironments (pH less than 7, greater than 4) to form an amine and analdehyde or a ketone are used in a variety of molecules and polymersthat include peptides, lipids, and liposomes.

The present invention additionally provides for the use of polymerscontaining silicon-oxygen-carbon linkages (either in the main chain ofthe polymer or in a side chain of the polymer) that are labile underacidic conditions. Organosilanes have long been utilized as oxygenprotecting groups in organic synthesis due to both the ease inpreparation (of the silicon-oxygen-carbon linkage) and the facileremoval of the protecting group under acidic conditions. For example,silyl ethers and silylenolethers, both posses such a linkage.Silicon-oxygen-carbon linkages are susceptible to hydrolysis underacidic conditions forming silanols and an alcohol (or enol). Thesubstitution on both the silicon atom and the alcohol carbon can affectthe rate of hydrolysis due to steric and electronic effects. This allowsfor the possibility of tuning the rate of hydrolysis of thesilicon-oxygen-carbon linkage by changing the substitution on either theorganosilane, the alcohol, or both the organosilane and alcohol tofacilitate the desired affect. In addition, charged or reactive groups,such as amines or carboxylate, may be linked to the silicon atom, whichconfers the labile compound with charge and/or reactivity.

The present invention additionally provides for the use of polymerscontaining silicon-nitrogen (silazanes) linkages (either in the mainchain of the polymer or in a side chain of the polymer) that aresusceptible to hydrolysis. Hydrolysis of a silazane leads to theformation of a silanol and an amine. Silazanes are inherently moresusceptible to hydrolysis than is the silicon-oxygen-carbon linkage,however, the rate of hydrolysis is increased under acidic conditions.The substitution on both the silicon atom and the amine can affect therate of hydrolysis due to steric and electronic effects. This allows forthe possibility of tuning the rate of hydrolysis of the silizane bychanging the substitution on either the silicon or the amine tofacilitate the desired affect.

The present invention additionally provides for the use of polymerscontaining silicon-carbon linkages (either in the main chain of thepolymer or in a side chain of the polymer) that are susceptible tohydrolysis. For example, arylsilanes, vinylsilanes, and allylsilanes allposses a carbon-silicon bond that is susceptible to hydrolysis.

C) Very pH-Labile Bonds

To construct labile molecules, one may construct the molecule with bondsthat are inherently labile such as disulfide bonds, diols, diazo bonds,ester bonds, sulfone bonds, acetals, ketals, enol ethers, enol esters,imines, imminiums, and enamines. In addition, one may construct apolymer in such a way as to put reactive groups, i.e. electrophiles andnucleophiles, in close proximity so that reaction between the functiongroups is more rapid than if the reactive groups are not in closeproximity. Examples include having carboxylic acid derivatives (acids,esters, amides) and alcohols, thiols, carboxylic acids or amines in thesame molecule reacting together to make esters, thiol esters, acidanhydrides or amides.

An example of the construction of labile molecules containing labilebonds is the use of the acid labile enol ether bond. The enol ether isan ether, a molecule containing a —C—O—C— linkage, in which one of thecarbons bonded to oxygen is sp2 hybridized and bonded to another carbon,i.e. an enol. Enols are unstable and rapidly convert to the carbonyl,i.e. the ketone or aldehyde. Enol ethers are stable, relative to enols,but under acidic aqueous conditions convert to alcohol and ketone oraldehyde. Depending on the structures of the carbonyl compound formedand the alcohol release, enol hydrolysis can be very pH-labile. Ingeneral, hydrolysis to form ketones is much faster than the rate ofconversion to aldehydes. For example the rate of hydrolysis of ethylisopropenyl ether to form ethanol and acetone is ca. 3600 times fasterthan the hydrolysis of ethyl trans-propenyl ether to form ethanol andpropanal.

Cleavage of an Enol Ether.

There are two relatively facile methods for the synthesis ofketone-generation enol ether, although the generation of enol ethers isnot limited to these methods and one skilled in the art may find more.One method, metal-liquid ammonia reduction of aromatic compounds, suchas phenol ethers, results in the reduction of one carbon-carbon doublebond to produce a diene (Birch A. J. J. Chem. Soc. 1946, 593). Anothermethod is the elimination of β-halogen ethers (where chloride, fluoride,bromide, and iodide are halogens) under basic conditions.

Two synthetic strategies for the generation of enol ethers

An advantage of these methods is that the labile enol ether is producedfrom relatively stable ethers. This stability of starting materialenables one to construct the labile molecule under conditions where itis not labile and then produce the labile enol ether linkage. Usingsuitable β-haloethers, both methods produce enol ethers that hydrolyzeinto ketones, which enable one to construct very pH-labile bonds. Forexample analogs of ethyl isopropenyl ether, which may be synthesizedfrom β-haloethers, have half-lives of roughly 2 minutes at pH 5 (Kresge,A. J.; Sagatys, D. S.; Chen, H. L. J. Am. Chem. Soc. 1977, 99, 7228). Afacile method for the production of a polymer containing isoproprenylether is the elimination of polyepichlorohydrin under basic conditions(Nishikubo, T., Iiazawa, T., Sugarwara, Y., and Shimokawa, T. J. Polym.Sci., Polym Chem. Ed. 1986, 24, 1097.) It has been shown (Perez, M.,Ronda, J. C., Reina, J. A., Serra, A. Polymer 1998, 39, 3885.) thatreaction of epichlorohydrin with phenolate salts is a competitionbetween substitution, to form the phenol ether, and elimination to formthe enol ether. To, illustrate the use of elimination of β-haloethers toconstruct enol ether-containing polyions, we reacted polyepichlorohydrinwith the tetrabutylammonium disalt of para-hydroxyphenylacetic acid. Theproduct was a polyanion, due to the substitution reaction, which hadenol ether functional groups. This polyanion's ability to from complexeswith polyallylamine was lost upon acidification. In addition this enoleither is very pH-labile: measurement of the rate of hydrolysis of theenol ether group by UV spectroscopy revealed a hydrolysis with ahalf-life of 37 minutes at pH 5.

Analogs of ethyl cyclohexenyl ether, which may be synthesized fromphenol ethers, have half-lives of roughly 14 minutes at pH 5 (Kresge, A.J.; Sagatys, D. S.; Chen, H. L. J. Am. Chem. Soc. 1977, 99, 7228). Toillustrate this approach to construct enol ethers, we synthesizedglycolic acid ethoxylate(4 units) 4-tert-buty-1,4-cyclohexadiene etherby metal-liquid ammonia reduction of glycolic acid ethoxylate(4 units)4-tert-buty-phenyl ether, which is a phenol ether. The hydrolysis ofthis enol ether is very acid labile. The half-life of the hydrolysis ofthis enol ether -containing surfactant was 40 minutes at pH 5.

D. Extremely pH-Labile Bonds

An illustrative embodiment of the present invention, in which proximityof reactive groups confers lability, is shown by the conversion of amineto amides with anhydrides. Reaction of an amine with an anhydrideresults in the formation of an amide and a carboxylic acid. As is thecase with all chemical reactions, this coupling of amine and anhydrideis, in theory, reversible. However, as is the case for many chemicalreactions, the reverse reaction (between a carboxylic acid and amide toform an anhydride and amine) is so unfavorable that the reaction betweenan amine and an anhydride is considered irreversible. Exceptions to thisirreversibility are observed when the anhydride is a cyclic anhydridesuch that the formed amide and acid are in the same molecule, an amideacid. Placement of both reactive groups (amide and carboxylic acid) inthe same molecule accelerates their reaction such that amine-anhydridereactivity becomes functionally reversible. For example, the product ofsuccinic anhydride and a primary amine, a succinamic acid, reverse backto amine and anhydride 10,000 times faster than the products betweennoncyclic anhydride and a primary amine. In particular, the product ofprimary amines with maleic anhydride and maleic anhydride derivatives,maleamic acids, revert back to amine and anhydride with amazing speed,1×10⁹ to 1×10¹³ times faster than its noncyclic analogues (Kirby, A J.J. Adv. Phys. Org Chem. 1980, 17, 183)

Reaction of an Amine and an Anhydride to Form an Amide Acid.

The amide acid that converts to amine and anhydride is the protonatedacid, not the deprotonated carboxylate. For this reason, cleavage of theamide acid to form amine and anhydride is pH-dependent. ThispH-dependent reactivity can be exploited to form reversible pH-sensitivelinkers. Linkers, or spacer molecules, are used to conjugate passengermolecules and carrier molecules, which increase the transport anddelivery of passenger molecules. Specifically, cis-aconitic acid is usedas such a pH-sensitive linker molecule. The γ-carboxylate is firstcoupled to a carrier molecule, a molecule that assists in delivery suchas an interaction modifier or a targeting ligand. In a second step,either the α or β carboxylate is coupled to a passenger molecule, suchas a biologically active compound, to form a pH-sensitive coupling ofpassenger and carrier molecules. An estimation of the kinetics ofcleavage between passenger and carrier reveals that at pH 5 thehalf-life of cleavage is between 8 and 24 hours (Blattler, W. A.;Kuenzi, B. S.; Lambert, J. M.; Senter, P. D. Biochemistry, 1985, 24,1517–1524).

Structures of Cis-Aconitic Anhydride and Maleic Anhydride.

The pH at which cleavage occurs is controlled by the addition ofchemical constituents to the labile moiety. The rate of conversion ofmaleamic acids to amines and maleic anhydrides is strongly dependent onsubstitution (R₂ and R₃) of the maleic anhydride system. When R₂ ismethyl (from citraconic anhydride and similar in substitution tocis-aconitic anhydride) the rate of conversion is 50-fold higher thanwhen R₂ and R₃ are hydrogen (derived from maleic anhydride). When thereare alkyl substitutions at both R₂ and R₃ (e.g.,2,3-dimethylmaleicanhydride) the rate increase is dramatic, 10000-foldfaster than maleic anhydride. Indeed, modification of the polycationpoly-L-lysine with 2,3-dimethylmaleic anhydride to form the polyanionic2,3-dimethylmaleamic poly-L-lysine, followed by incubation at acidic pHresulted in loss of 2,3-dimethylmaleic and return of the polycationpoly-L-lysine. The half-life of this conversion was between 4 and 10minutes at pH 5. This shows that conversion of 2,3-dimethylmaleamicacids (derived from the reaction between 2,3-dimethylmaleic anhydrideand amines at basic pH), to amines and 2,3-dimethylmaleic anhydride atacidic pH is extremely labile. It is postulated that this increase inrate for 2,3-dimethylmaleamic acids is due to the steric interactionsbetween the two methyl groups which increases the interaction betweenamide and carboxylate and thereby increases the rate of conversion toamine and anhydride. Therefore, it is anticipated that if R₂ and R₃ aregroups larger than hydrogen, which includes any conceivable group, therate of amide-acid conversion to amine and anhydride will be faster thanif R₂ and/or R₃ are hydrogen. One would expect that 2,3-diethylmaleamicacids to cleave faster than ethylmaleamic acids and so forth. Inaddition, we synthesized 2-propionic-3-methylmaleic anhydride and foundthat the rate of 2-propionic-3-methylmaleamic acid cleavage was the sameas that for 2,3-dimethylmaleamic acids.

Another method for the production of rapidly cleaved pH-sensitivederivatives of maleic anhydride is to react the anhydride with analcohol or thiol to form an acid ester or acid thioester.

II. Polymers with pH-Labile Bonds

Polymers with labile bonds may have the following generalizedstructures: A-B-A where A is a monomer and B is a pH-labile linkage,A-B-C where A is a monomer and B is a pH-labile linkage and C is aninteraction modifier. The modifying group may confer the polymer with avarieties of new characteristics such as a change in charge (e.g.cationic, anionic), cell targeting capabilities (e.g. nuclearlocalization signals), hydrophilicity (e.g. polyethyleneglycol,saccharides, and polysaccharides), and hydrophobicity (e.g. lipids anddetergents). The labile group may be added to the polymer during polymersynthesis or the labile group may be added to the polymer afterpolymerization has occurred.

The present invention provides a wide variety of polymers with labilegroups that find use in the delivery systems of the present invention.The labile groups are selected such that they undergo a chemicaltransformation (e.g., cleavage) in physiological conditions, that is,when introduced into a specific, inherent intra or extracellularenvironment (e.g., the lower pH conditions of an endosome, or theextracellular environment of a cancerous tumor). In addition, thechemical transformation may also be initiated by the addition of acompound. The conditions under which a labile group will undergotransformation can be controlled by altering the chemical constituentsof the molecule containing the labile group. For example, addition ofparticular chemical moieties (e.g., electron acceptors or donors) nearthe labile group can effect the particular conditions (e.g., pH) underwhich chemical transformation will occur. The present invention providesassays for the selection of the desired properties of the labile groupfor any desired application. A labile group is selected based upon itshalf-life and is included in a polymer. The polymer is then complexedwith the biologically active compounds and an in vitro or in vivo assayis used to determine whether the compound's activity is affected.

A. pH-Labile Linkages within pH-Labile Polymers

The pH-labile bond may either be in the main-chain or in the side chain.If the pH-labile bond occurs in the main chain, then cleavage of thelabile bond results in a decrease in polymer length. If the pH-labilebond occurs in the side chain, then cleavage of the labile bond resultsin loss of side chain atoms from the polymer.

An example of a pH-labile bond in the side chain of a polymer is2,3-dimethylmaleamic poly-L-lysine, which is formed by the reaction ofpoly-L-lysine with 2,3-dimethylmaleic anhydride under basic conditions.The modification of the poly-L-lysine is in the side chain andconversion of the 2,3-dimethylmaleamic poly-L-lysine to poly-L-lysineand 2,3-dimethylmaleic anhydride under acid conditions does not resultin a cleavage of the polymer main, but in a cleavage of the side chain.

An example of a silicon-oxygen-carbon pH-labile bond in the side chainof the polymer is the polymer formed from the reaction of poly-L-serineand 3-aminopropyltrimethoxysilane in DMF. The ratio of3-aminopropyl-trimethoxysilane per serine monomer units may be changedresulting in differing amounts of silylether formation. Hydrolysis ofthe polymer under acidic conditions regenerates the poly-L-serine and asilanol.

An example of a pH-labile bond in the main chain of the polymer isdi-(2-methyl-4-hydroxymethyl(succinic semialdehydeester)-1,3-dioxolane)-1,4-benzene:1,4-bis(3-aminopropyl)piperazinecopolymer (1:1) (MC208) prepared from the reaction ofdi-(2-methyl-4-hydroxymethyl(succinic semialdehydeester)-1,3-dioxolane)-1,4-benzene and: 1,4-bis(3-aminopropyl)piperazine.The resulting polymer (containing imines) can be reduced in the presenceof NaCNBH₃ to afford the secondary amine containing copolymer (MC301)which retains the pH-lability of the parent polymer, through ketalfunctional groups. Both polymers contain a substituted 1,3-dioxolanering system, ketal, which upon exposure to acidic environmentshydrolyzes to a ketone and diol.

B. Polymerization Processes to Form the pH-Labile Polymers

There are a number of polymerization processes that can be utilized withthe present invention. For example, the polymerization can be chain orstep. This classification description is more often used that theprevious terminology of addition and condensation polymer. “Moststep-reaction polymerizations are condensation processes and mostchain-reaction polymerizations are addition processes” (M. P. StevensPolymer Chemistry: An Introduction New York Oxford University Press1990). Template polymerization can be used to form polymers fromdaughter polymers.

1. Step Polymerization: In step polymerization, the polymerizationoccurs in a stepwise fashion. Polymer growth occurs by reaction betweenmonomers, oligomers and polymers. No initiator is needed since there isthe same reaction throughout and there is no termination step so thatthe end groups are still reactive. The polymerization rate decreases asthe functional groups are consumed.

Typically, step polymerization is done either of two different ways. Oneway, the monomer has both reactive functional groups (A and B) in thesame molecule so that A-B yields -[A-B]-Or the other approach is to havetwo bifunctional monomers. A—A+B—B yields -[A—A-B—B]-Generally, thesereactions can involve acylation or alkylation. Acylation is defined asthe introduction of an acyl group (—COR) onto a molecule. Alkylation isdefined as the introduction of an alkyl group onto a molecule.

If functional group A is an amine then B can include, but is not limitedto, an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide,sulfonyl chloride, aldehyde (including formaldehyde and glutaraldehyde),ketone, epoxide, carbonate, imidoester, carboxylate activated with acarbodiimide, alkylphosphate, arylhalides (difluoro-dinitrobenzene),anhydride, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium. In other termswhen function A is an amine then function B can be acylating oralkylating agent or amination agent.

If functional group A is a thiol, sulfhydryl, then function B caninclude, but is not limited to, an iodoacetyl derivative, maleimide,aziridine derivative, acryloyl derivative, fluorobenzene derivatives, ordisulfide derivative (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid {TNB} derivatives).

If functional group A is carboxylate then function B can include, but isnot limited to, a diazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

If functional group A is an hydroxyl then function B can include, but isnot limited to, an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used.

If functional group A is an aldehyde or ketone then function B caninclude, but is not limited to, an hydrazine, hydrazide derivative,amine (to form a imine or iminium that may or may not be reduced byreducing agents such as NaCNBH₃) or hydroxyl compound to form a ketal oracetal.

Yet another approach is to have one bifunctional monomer so that A—Aplus another agent yields-[A—A]-. If function A is a thiol, sulfhydryl,group then it can be converted to disulfide bonds by oxidizing agentssuch as iodine (I₂) or NaIO₄ (sodium periodate), or oxygen (O₂).Function A can also be an amine that is converted to a thiol,sulfhydryl, group by reaction with 2-Iminothiolate (Traut's reagent)which then undergoes oxidation and disulfide formation. Disulfidederivatives (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) can also be used to catalyze disulfide bondformation.

Functional group A or B in any of the above examples could also be aphotoreactive group such as aryl azide (including halogenated arylazide), diazo, benzophenone, alkyne or diazirine derivative.

Reactions of the amine, hydroxyl, thiol, sulfhydryl, carboxylate groupsyield chemical bonds that are described as amide, amidine, disulfide,ethers, esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondary amine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone.

2. Chain Polymerization: In chain-reaction polymerization, growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.

Monomers containing (but not limited to) vinyl, acrylate, methacrylate,acrylamide, methacrylamide groups can undergo chain reaction which canbe radical, anionic, or cationic. Chain polymerization can also beaccomplished by cycle or ring opening polymerization. A number ofdifferent types of free radical initiators could be used that includeperoxides, hydroxy peroxides, and azo compounds such as2,2′-Azobis(-amidinopropane)dihydrochloride (AAP).

C. Types of Monomers for Incorporation into pH-Labile Polymers and Typesof pH-Labile Polymers

A wide variety of monomers can be used in the polymerization processes.These include positive charged organic monomers such as amines, aminesalts, imidine, guanidine, imine, hydroxylamine, hydrazine, heterocycleslike imidazole, pyridine, morpholine, pyrimidine, or pyrene. Polymersfrom such monomers includes, but are not limited to such examples aspoly-L-lysine, polyethylenimine (linear and branched), andpolyallylamine. The amines could be pH-sensitive in that the pKa of theamine is within the physiologic range of 4 to 8. Specific pH-sensitiveamines include spermine, spermidine,N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

In addition negatively charged monomers such as sulfates, sulfonates,carboxylates, and phosphates may be used to generated polyanionicpolymers. Examples of these polyanions include, but are not limited to,nucleic acids, polysulfonylstyrene, and heparin sulfate. Also,amine-containing polycations may be converted to polyanions by reactionwith cyclic anhydrides such as succinic anhydride and glutaric anhydrideto form glutarylated and succinylated polymers which are polyanionic.Examples of these polyanions include, but are not limited to,succinylated and glutarylated poly-L-lysine, and succinylated andglutarylated polyallylamine.

Monomers can also be hydrophobic, hydrophilic or amphipathic.

Monomers can also be intercalating agents such as acridine, thiazoleorgange, or ethidium bromide. Monomers can also contain chemicalmoieties that can be modified before or after the polymerizationincluding (but not limited to) amines (primary, secondary, andtertiary), amides, carboxylic acid, ester, hydroxyl, hydrazine, alkylhalide, aldehyde, and ketone.

The pH-labile polymer can be a polyion, polycation, polyanion,zwitterionic polymers, and neutral polymers. It can also contain achelator and be a polychelator.

D. Other Components of the Monomers and Polymers

The polymers may include other groups that increase their utility. Thesegroups can be incorporated into monomers prior to polymer formation orattached to the polymer after its formation. These groups include, butare not limited to: targeting groups and signals (e.g, cell receptor,nuclear targeting signals), membrane active compounds, reporter ormarker molecules, spacers, steric stabilizers, chelators, polycations,polyanions, and polymers.

III. Polymers Containing Several Membrane Active Compounds

The present invention specifies polymers containing more than twomembrane active compounds. In one embodiment, the membrane activecompounds are grafted onto a preformed polymer to form a comb-typepolymer. For example, both the membrane active peptides melittin and KL₃contain only one carboxylate, which is at the carboxy terminus.Therefore, activation of the peptides with carboxy-activating agentssuch as carbodiimides will react with only one group. If this activationis done in the presence of an excess of an amine, then one may obtainselective amide formation. In particular, if the activation is done inthe presence of a polyamine, one would obtain selective coupling of thepeptide to the polyamine. This method of coupling of a membrane activepeptide to a polyamine was accomplished for the coupling of peptides KL₃and melittin to polyamines polyallylamine and poly-L-lysine. In eachcase, the membrane activity, as judged by hemolysis, was retained and,in case of KL₃, was improved after attachment to the polycation.

In another embodiment, the membrane active compounds are incorporatedinto the polymer by chain or step polymerization processes. For example,an acryloyl group at the N-terminus of a peptide allows one to form apolyacrylamide polymer with peptide side chains (O'Brien-Simpson, N. M.,Ede, N. J., Brown, L. E., Swan, J., Jackson, D. C J. Am. Chem. Soc.1997, 119, 1183). N-acryloyl KL3 was synthesized and polymerized andfound to retain the activity of monomeric KL3, but was able to formparticles with DNA.

IV. Membrane Active Compounds Containing Labile Bonds

The invention specifies compounds that are of the general structure:A-B-C wherein A is a membrane active compound, B is a labile linkage,and C is a compound that inhibits the membrane activity of compound A. Amembrane active compound is defined within the Definitions Section andincludes membrane active peptides.

The term labile linkage is defined above and includes pH-labile bondssuch as, acetals, ketals, enol ethers, enol esters, enamines, andimines. It also includes extremely pH-labile bonds such as2,3-disubstituted maleamic acids and very pH-labile bonds such as enolethers.

Preferred embodiments include 2,3-dimethylmaleamic-mellitin,2-propionic-3-methylmaleamic melittin, 2-propionic-3-methylmaleamic KL3,and 2,3-dimethylmaleamic-melittin, which are membrane inactive compoundsthat become membrane active under acidic conditions.

The disulfide linkage (RSSR′) may be used within bifunctional molecules.The reversibility of disulfide bond formation makes them useful toolsfor the transient attachment of two molecules. Disulfides have been usedto attach a bioactive compound and another compound (Thorpe, P. E. J.Natl. Cancer Inst. 1987, 79, 1101). The disulfide bond is reducedthereby releasing the bioactive compound. Disulfide bonds may also beused in the formation of polymers (Kishore, K., Ganesh, K. in Advancesin Polymer Science, Vol. 21, Saegusa, T. Ed., 1993).

In another embodiment, the invention includes compositions containingbiologically active compounds and compounds of the general structure:A-B-C wherein A is a membrane active compound, B is a labile linkage,and C is a compound that inhibits the membrane activity of compound A.The biologically active compounds include pharmaceutical drugs, nucleicacids and genes. In yet another embodiment, these compounds that are ofthe general structure—A-B-C wherein A is a membrane active compound, Bis a labile linkage, and C is a compound that inhibits the membraneactivity of compound A- are used to deliver biologically activecompounds that include pharmaceutical drugs, nucleic acids and genes. Inone specific embodiment, these A-B-C compounds are used to delivernucleic acids and genes to muscle (skeletal, heart, respiratory,striated, and non-striated), liver (hepatocytes), spleen, immune cells,gastrointestinal cells, cells of the nervous system (neurons, glial, andmicroglial), skin cells (dermis and epidermis), joint and synovialcells, tumor cells, kidney, cells of the immune system (dendiritic, Tcells, B cells, antigen-presenting cells, macrophages), exocrine cells(pancreas, salivary glands), prostate, adrenal gland, thyroid gland, eyestructures (retinal cells), and respiratory cells (cells of the lung,nose, respiratory tract). Upon cleavage of B, membrane activity isrestored to compound A. This cleavage occurs in certain tissue, organ,and sub-cellular locations that are controlled by the microenvironmentof the location and also by the addition of exogenous agents. Deliverycan be accomplished by direct intraparenchymal injections (into theparenchyma of a tissue) or by intravascular conditions. Intravascularconditions also include conditions under which the permeability of thevessel is increased and when the injection is leads to increasedintravascular pressure.

V. Mixtures of Membrane Active Compounds and Labile Compounds

In addition, the invention is a composition of matter that includes amembrane active compound and a labile compound. In one embodiment, thelabile compound inhibits the membrane activity of the membrane activecompound. Upon chemical modification of the labile compound, membraneactivity is restored to the membrane active compound. This chemicalmodification occurs in certain tissue, organ, and sub-cellular locationsthat are controlled by the microenvironment of the location and also bythe addition of exogenous agents. In one embodiment the chemicalmodification involves the cleavage of the polymer. In one embodiment,the membrane active compound and the inhibitory labile compound arepolyions and are of opposite charge. For example, the membrane activecompound is a polycation and the inhibitory labile compound is apolyanion, or the membrane active compound is a polyanion and theinhibitory labile compound is a polycation.

In another embodiment, the invention includes compositions containingbiologically active compounds, a membrane active compound and a labilecompound. Upon chemical modification of the labile compound, membraneactivity is restored to the membrane active compound. This chemicalmodification occurs in certain tissue, organ, and sub-cellular locationsthat are controlled by the microenvironment of the location and also bythe addition of exogenous agents. In one embodiment the chemicalmodification involves the cleavage of the polymer. In one specificembodiment, these compositions containing biologically active compounds,a membrane active compound and a labile compound are used to delivernucleic acids and genes to muscle (skeletal, heart, respiratory,striated, and non-striated), liver (hepatocytes), spleen, immune cells,gastrointestinal cells, cells of the nervous system (neurons, glial, andmicroglial), skin cells (dermis and epidermis), joint and synovialcells, tumor cells, kidney, cells of the immune system (dendiritic, Tcells, B cells, antigen-presenting cells, macrophages), exocrine cells(pancreas, salivary glands), prostate, adrenal gland, thyroid gland, eyestructures (retinal cells), respiratory cells (cells of the lung, nose,respiratory tract), and endothelial cells.

VI. Biologically Active Compounds Containing very and/or ExtremelypH-Labile Bonds

The invention specifies compounds of the following general structure:A-B-C wherein A is a biologically active compound such aspharmaceuticals, drugs, proteins, peptides, hormones, cytokines, enzymesand nucleic acids such as anti-sense, ribozyme, recombining nucleicacids, and expressed genes; B is a labile linkage that contains apH-labile bond such as amides of 2,3-dimethylmaleamic acid, enol ethers,enol esters, silyl ethers, and silyl enol ethers; and C is a compound.In one embodiment C is a compound that modifies the activity, function,delivery, transport, shelf-life, pharmacokinetics, blood circulationtime in vivo, tissue and organ targetting, and sub-cellular targeting ofthe biologically active compound A. In other embodiments, B is a labilelinkage that contains acetals, ketals, enol ethers, enol esters, amides,imines, imminiums, enamines, silyl ethers, or silyl enol ethers.

The invention also specifies that the labile linkage B is attached toreactive functional groups on the biologically active compound A. In yetanother embodiment, reactive functional groups are attached to nucleicacids. Specifically, aziridines, quinones, oxiranes, epoxides, nitrogenmustards, sulfur mustards, and halogen and carbon-containing compoundssuch as alkylhalides, halo-amines, alpha-halo amides, esters and acids,may be used to modify nucleic acids and thereby attach reactivefunctional groups.

VII. pH-Labile Amphipathic Compounds

In one specification of the invention, the pH-labile and very pH-labilelinkages and bonds are used within amphipathic compounds and detergents.The pH-labile amphipathic compounds can be incorporated into liposomesfor delivering biologically active compounds and nucleic acids to cells.The detergents can be used for cleaning purposes and for modifying thesolubility of biologically active compounds such as proteins. pH-labilesurfactants may be desirable for their reversible solubilization ofhydrophobic compounds in water and hydrophilic compounds in organicsolvents. For example, surfactants are necessary for the purification ofmembrane proteins; however, it is often difficult to separate membraneproteins and surfactants once the purification is complete. Labilesurfactants may also be more biodegradable and may reverse the formationof unwanted emulsions or foams.

For example the surfactant glycolic acid ethoxylate(4 units)4-tert-buty-phenyl ether was converted in the enol-ether-containingpH-labile surfactant glycolic acid ethoxylate(4 units)4-tert-buty-1,4-cyclohexadiene ether by ammonia-metal reduction of thephenyl group. The enol-ether bond links the hyrdrophilic portion of themolecule with the hydrophobic portion of the molecule, therefore,cleavage of the enol ether bond renders the amphiphilic surfactant intotwo separate molecules (one hydrophilic and one hydrophobic). Thehalf-life of enol ether cleavage was 40 minutes at pH 5. In likewisemanner, similar surfactants such as Triton X-100 may be converted intopH-labile surfactants.

I) Delivery Systems

In some embodiments of the present invention, the labile group (e.g.,ester, amide or thioester acid) is complexed with lipids and liposomesso that in acidic environments the lipids are modified and the liposomebecomes disrupted, fusogenic or endosomolytic. For example, the lipiddiacylglycerol is reacted with an anhydride to form an ester acid. Afteracidification in an intracellular vesicle the diacylglycerol reforms andis very lipid bilayer disruptive and fusogenic.

In preferred embodiments of the present invention the delivery systemscomprise polymers.

One of the several methods of nucleic acid delivery to the cells is theuse of DNA-polycation complexes. It has been shown that cationicproteins like histones and protamines or synthetic polymers likepolylysine, polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular delivery agents whilesmall polycations like spermine are ineffective.

In addition to the delivery of polynucleotides, other bioactivemolecules, such as proteins and small molecule drugs, may be deliveredusing a labile connection. Either through a direct modification of thebioactive molecule or through the formation of a complex with themolecule, which is itself labile.

EXAMPLES Example 1

Synthesis and Characterization of Labile Compounds

A) Synthesis of 2-propionic-3-methylmaleic anhydride(carboxydimethylmaleic anhydride or C-DM): To a suspension of sodiumhydride (0.58 g, 25 mmol) in 50 mL anhydrous tetrahydrofuran was addedtriethyl-2-phosphonopropionate (7.1 g, 30 mmol). After bubbling ofhydrogen gas stopped, dimethyl-2-oxoglutarate (3.5 g, 20 mmol) in 10 mLanhydrous tetrahydrofuran was added and stirred for 30 minutes. Water,10 mL, was then added and the tetrahydrofuran was removed by rotaryevaporation. The resulting solid and water mixture was extracted with3×50 mL ethyl ether. The ether extractions were combined, dried withmagnesium sulfate, and concentrated to a light yellow oil. The oil waspurified by silica gel chromatography elution with 2:1 ether:hexane toyield 4 gm (82% yield) of pure triester. The 2-propionic-3-methylmaleicanhydride then formed by dissolving of this triester into 50 mL of a50/50 mixture of water and ethanol containing 4.5 g (5 equivalents) ofpotassium hydroxide. This solution was heated to reflux for 1 hour. Theethanol was then removed by rotary evaporation and the solution wasacidified to pH 2 with hydrochloric acid. This aqueous solution was thenextracted with 200 mL ethyl acetate, which was isolated, dried withmagnesium sulfate, and concentrated to a white solid. This solid wasthen recrystallized from dichloromethane and hexane to yield 2 g (80%yield) of 2-propionic-3-methylmaleic anhydride.

B) Synthesis of 2,3-dioleoyldiaminopropionic ethylenediamine amide:2,3-diaminopropionic acid (1.4 gm, 10 mmol) and dimethylaminopyridine(1.4 gm 11 mmol) were dissolved in 50 mL of water. To this mixture wasadded over 5 minutes with rapid stirring oleoyl chloride (7.7 mL, 22mmol) of in 20 mL of tetrahydrofuran. After all of the acid chloride hadbeen added, the solution was allowed to stir for 30 minutes. The pH ofthe solution was 4 at the end of the reaction. The tetrahydrofuran wasremoved by rotary evaporation. The mixture was then partitioned betweenwater and ethyl acetate. The ethyl acetate was isolated, dried withmagnesium sulfate, and concentrated by rotary evaporation to yield ayellow oil. The 2,3-dioleoyldiaminopropionic acid was isolated by silicagel chromatography, elution with ethyl ether to elute oleic acid,followed by 10% methanol 90% methylene chloride to elute diamideproduct, 1.2 g (19% yield). The diamide (1.1 gm, 1.7 mmol) was thendissolved in 25 mL of methylene chloride. To this solution was addedN-hydroxysuccinimide (0.3 g. 1.5 eq) and dicyclohexylcarbodiimide (0.54g, 1.5 eq). This mixture was allowed to stir overnight. The solution wasthen filtered through a cellulose plug. To this solution was addedethylene diamine (1 gm, 10 eq) and the reaction was allowed to proceedfor 2 hours. The solution was then concentrated by rotary evaporation.The resulting solid was purified by silica gel chromatography elutionwith 10% ammonia saturated methanol and 90% methylene chloride to yieldthe triamide product 2,3-dioleoyldiaminopropionic ethylenediamine amide(0.1 gm, 9% yield). The triamide product was given the number MC213.

C) Synthesis of dioleylamideaspartic acid:N-(tert-butoxycarbonyl)-L-aspartic acid (0.5 gm, 2.1 mmol) was dissolvedin 50 mL of acetonitrile. To this solution was addedN-hydroxysuccinimide (0.54 gm, 2.2 eq) and was addeddicyclohexylcarbodiimide (0.54 g, 1.5 eq). This mixture was allowed tostir overnight. The solution was then filtered through a cellulose plug.This solution was then added over 6 hours to a solution containingoleylamine (1.1 g, 2 eq) in 20 mL methylene chloride. After the additionwas complete the solvents were removed by rotary evaporation. Theresulting solid was partitioned between 100 mL ethyl acetate and 100 mLwater. The ethyl acetate fraction was then isolated, dried by sodiumsulfate, and concentrated to yield a white solid. The solid wasdissolved in 10 mL of triflouroacetic acid, 0.25 mL water, and 0.25 mLtriisopropylsilane. After two hours, the triflouroacetic acid wasremoved by rotary evaporation. The product was then isolated by silicagel chromatography using ethyl ether followed by 2% methanol 98%methylene chloride to yield 0.1 gm (10% yield) of puredioleylamideaspartic acid, which was given the number MC303.

D) Synthesis of2,3-dimethylmaleamic poly-L-lysine: Poly-L-lysine (10 mg34,000 MW Sigma Chemical ) was dissolved in 1 mL of aqueous potassiumcarbonate (100 mM). To this solution was added 2,3-dimethylmaleicanhydride (100 mg, 1 mmol) and the solution was allowed to react for 2hr. The solution was then dissolved in 5 mL of aqueous potassiumcarbonate (100 mM) and dialyzed against 3×2 L water that was at pH8 withaddition of potassium carbonate. The solution was then concentrated bylyophilization to 10 mg/mL of 2,3-dimethylmaleamic poly-L-lysine.

E) Synthesis of dimethylmaleamic-melittin and dimethylmaleamic-pardaxin.Solid melittin or pardaxin (100 μg) was dissolved in 100 μL of anhydrousdimethylformamide containing 1 mg of 2,3-dimethylmaleic anhydride and 6μL of diisopropylethylamine.

F) Synthesis of dimethylmaleic derivatives (from alcohol-containing) anddimethylmaleamic derivatives (from amine-containing) of lipids: To asolution of 1 mg of lipid (either MC 213, MC 303,phosphatidylethanolamine dioleoyl (DOPE), or 1,2-dioleoylglycerol (DOG))in chloroform (0.1 mL) is added 10 mg of 2,3-dimethylmaleic anhydrideand 82 mg of diisopropylethyl amine. The solution is allowed to incubateat room temperature for 1 hour before testing for activity.

G) Synthesis of2-propionic-3-methylmaleic derivatives (fromalcohol-containing) and 2-propionic-3-methylmaleamic derivatives (fromamine-containing) of lipids: To a solution of 1 mg of lipid (either MC213, MC 303, phosphatidylethanolamine dioleoyl (DOPE), or1,2-dioleoylglycerol (DOG)) in chloroform (0.1 mL) is added 10 mg of2-propionic-3-methylmaleic anhydride and 82 mg of diisopropylethylamine. The solution is allowed to incubate at room temperature for 1hour before testing for activity.

H) Synthesis of adducts between peptide and poly-L-lysine adducts: To asolution of poly-L-lysine (10 mg, 0.2 μmol) and peptides KL₃ or melittin(2 μmol) is added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (20 μmol). For the peptide KL₃, the reaction is performedin 2 mL of water. For the peptide melittin, the reaction is performed ina solution of 1 mL water and 1 mL triflouroethanol. The reaction isallowed to proceed overnight before placement into a 12,000 molecularweight cutoff dialysis bag and dialysis against 4×2 liters over 48hours. The amount of coupled peptide is determined by the absorbance ofthe tryptophan residue at 280 nm, using an extinction coefficient of5690 cm⁻¹M⁻¹ (Gill, S. C. and von Hippel, P. H. Analytical Biochemistry(1989) 182, 319–326). The conjugate of melittin and poly-L-lysine wasdetermined to have 4 molecules of melittin per molecule of poly-L-lysineand is referred to as mel-PLL. The conjugate of KL₃ and poly-L-lysinewas determined to have 10 molecules of KL₃ per molecule of poly-L-lysineand is referred to as KL₃-PLL.

I) Synthesis of adducts between peptide and polyallylamine adducts: To asolution of polyallylamine (10 mg, 0.2 μmol) and peptides KL₃ ormelittin (2 μmol) is added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (20 μmol). For the peptide KL₃, the reaction is performedin 2 mL of water. For the peptide melittin, the reaction is performed ina solution of 1 mL water and 1 mL triflouroethanol. The reaction isallowed to proceed overnight before placement into a 12,000 molecularweight cutoff dialysis bag and dialysis against 4×2 liters over 48 hoursto remove uncoupled peptide. The amount of coupled peptide is determinedby the absorbance of the tryptophan residue at 280 nm, using anextinction coefficient of 5690 cm⁻¹M⁻¹ (Gill, S. C. and von Hippel, P.H. Analytical Biochemistry (1989) 182, 319–326). The conjugate melittinand polyallylamine was determined to have 4 molecules of melittin permolecule of polyallylamine and is referred to as mel-PAA. The conjugateof KL₃ and polyallylamine was determined to have 10 molecules of KL₃ permolecule of polyallylamine and is referred to as KL₃-PAA.

J) Synthesis of polyethyleneglycol methyl ether2-propionic-3-methylmaleate (CDM-PEG): To a solution of2-propionic-3-methylmaleic anhydride (30 mg, 0.16 mmol) in 5 mLmethylene chloride was added oxalyl chloride (200 mg, 10 eq) anddimethylformamide (1 μL). The reaction was allowed to proceed overnightat which time the excess oxalyl chloride and methylene chloride wereremoved by rotary evaporation to yield the acid chloride, a clear oil.The acid chloride was dissolved in 1 mL of methylene chloride. To thissolution was added polyethyleneglycol monomethyl ether, molecular weightaverage of 5,000 (815 mg, 1 eq) and pyridine (20 μL, 1.5 eq) in 10 mL ofmethylene chloride. The solution was then stirred overnight. The solventwas then removed and the resulting solid was dissolved into 8.15 mL ofwater.

K) General procedure for the reaction of mel-PAA, KL₃-PAA, mel-PLL, andKL₃-PLL with dimethylmaleic anhydride and 2-propionic-3-methylmaleicanhydride: Peptide-polycation conjugates (10 mg/mL) in water werereacted with a ten-fold weight excess of dimethylmaleic anhydride and aten-fold weight excess of potassium carbonate. Analysis of the aminecontent after 30 by addition of peptide solution to 0.4 mMtrinitrobenzene sulfonate and 100 mM borax revealed no detectableamounts of amine.

L) Synthesis of glycolic acid ethoxylate(4 units)4-tert-buty-1,4-cyclohexadiene ether (an enolether containingdetergent): To a solution of glycolic acid ethoxylate(4 units)4-tert-buty-phenyl ether (100 mg, 0.26 mmol), t-butylalcohol (10 mL),and tetrahydrofuran (10 mL) was condensed liquid anhydrous ammonia (20mL) at −78° C. To this solution was added sodium metal (100 mg, 16 eq).The solution turned dark blue and was stirred for 4 hours during whichtime the blue color remained. The solution was then quenched by theaddition of ammonium chloride (220 mg, 16 eq). The ammonia was allowedto evaporate overnight. The mixture was then partitioned between 10 mLof water and 10 mL ethyl ether. The water layer was isolated and usedfor kinetic studies.

M) Synthesis ofpoly(oxy-1-para-aceticphenoxymethylethylene-co-oxy-1-methylethylene): Topara-hydroxyphenylacetic acid (0.115 gm, 0.75 mmol) was added a 1 Msolution of tetrabutylammonium hydroxide in methanol. The methanol wasthen removed by rotary evaporation to yield an oil. To this was added 5mL of tetrahydrofuran and polyepichlorohydrin (0.046 gm, 0.6 mg). Thesolution was then heated to 60° C. for 16 hours. The solution was thenplaced into dialysis tubing (12,000 molecular weight cutoff) anddialyzed against 2×1 L of water that was pH 9 with addition of potassiumcarbonate. A portion of this solution was filtered through a 0.2 μmnylon syringe filter, and then lyophilized to determine itsconcentration. This solution was used for particle formation andhydrolysis studies.

N) Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer: Toa solution of N,N′-Bis(t-BOC)-L-cystine (85 mg, 0.15 mmol) in ethylacetate (20 mL) was added N,N′-dicyclohexylcarbodiimide (108 mg, 0.5mmol) and N-hydroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, thesolution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (54 μL, 0.25 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h. The ethylacetate was then removed by rotary evaporation and the resulting solidwas dissolved in trifluoroacetic acid (9.5 mL), water (0.5 mL) andtriisopropylsilane (0.5 mL). After 2 h, the trifluoroacetic acid wasremoved by rotary evaporation and the aqueous solution was dialyzed in a15,000 MW cutoff tubing against water (2×21) for 24 h. The solution wasthen removed from dialysis tubing, filtered through 5 μM nylon syringefilter and then dried by lyophilization to yield 30 mg of polymer.

Synthesis of Acid Labile Monomers:

O) Synthesis of Di-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene(MC 216): To a solution of diacetylbenzene (2.00 g, 12.3 mmol, AldrichChemical Company) in toluene (30.0 mL), was added glycerol (5.50 g, 59.7mmol, Acros Chemical Company) followed by p-toluenesulfonic acidmonohydrate (782 mg, 4.11 mmol, Aldrich Chemical Company). The reactionmixture was heated at reflux for 5 hrs with the removal of water byazeotropic distillation in a Dean-Stark trap. The reaction mixture wasconcentrated under reduced pressure, and the residue was taken up inEthyl Acetate. The solution was washed 1×10% NaHCO₃, 3×H₂O, 1× brine,and dried (MgSO₄). Following removal of solvent (aspirator), the residuewas purified by flash chromatography on silica gel (20×150 mm, CH₂Cl₂eluent) to afford 593 mg (16% yield) ofdi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. Molecular ioncalculated for C₁₆H₂₂O₆ 310. found m+1/z 311.2. 300 MHz NMR (CDCl₃, ppm)δ 7.55–7.35 (4H, m) 4.45–3.55 (10H, m) 1.65 (6 H, brs).

P) Di-(2-methyl-4-hydroxymethyl(succinic semialdehydeester)-1,3-dioxolane)-1,4-benzene (MC 211): To a solution of succinicsemialdehyde (150 mg, 1.46 mmol, Aldrich Chemical Company) anddi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (150 mg, 480μmol) in CH₂Cl₂ (4 mL) was added dicyclohexylcarbodiimide (340 mg, 1.65mmol, Aldrich Chemical Company) followed by a catalytic amount of4-dimethylaminopyridine. The solution was stirred for 30 min andfiltered. Following removal of solvent (aspirator), the residue waspurified by flash chromatography on silica gel (20×150 mm, CH₂Cl₂eluent) to afford 50 mg (22%) of di-(2-methyl-4-hydroxymethyl(succinicsemialdehyde ester)-1,3-dioxolane)-1,4-benzene. Molecular ion calculatedfor C₂₄H₃₀O₁₀ 478.0. found m+1/z 479.4.

Q) Di-(2-methyl-4-hydroxymethyl(glyoxilic acidester)-1,3-dioxolane)-1,4-benzene (MC225): To a solution of glyoxylicacid monohydrate (371 mg, 403 μmol, Aldrich Chemical Company) anddi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (500 mg, 161μmol) in dimethylformamide (8 mL) was added dicyclohexylcarbodiimide(863 mg, 419 μmol, Aldrich Chemical Company). The solution was stirredfor 30 min and filtered. Following removal of solvent (aspirator), theresidue was purified by flash chromatography on silica gel (20×150 mm,ethylacetate/Hexanes (1:2.3 eluent) to afford 58 mg (10%) ofdi-(2-methyl-4-hydroxymethyl(glyoxylic acidester)-1,3-dioxolane)-1,4-benzene.

R) Di-(2-methyl-4-aminomethyl-1,3-dioxolane)-1,4-benzene (MC372). To asolution of 1,4-diacetylbenzene (235 mg, 1.45 mmol, Aldrich ChemicalCompany) in toluene (15.0 mL) was added 3-amino-1,2-propanediolprotected as the FMOC carbamide (1.0 g, 3.2 mmol), followed by acatalytic amount of p-toluenesulfonic acid monohydrate (Aldrich ChemicalCompany). The reaction mixture was heated at reflux for 16 hrs with theremoval of water by azeotropic distillation in a Dean-Stark trap. Thereaction mixture was cooled to room temperature, partitioned intoluene/H₂O, washed 1×10% NaHCO₃, 3×H₂O, 1× brine, and dried (MgSO₄).The extract was concentrated under reduced pressure and crystallized(methanol/H₂O). The protected amine ketal was identified in thesupernatant, which was concentrated to afford 156 mg product. The freeamine was generated by treating the ketal with piperidine indichloromethane for 1 hr.

S) Di-(2-methyl-4-hydroxymethyl(glycineester)-1,3-dioxolane)-1,4-benzene (MC373): To a solution of FMOC-Glycine(690 mg, 2.3 mmol, NovaBiochem) in dichloromethane (4.0 mL) was addeddicyclohexylcarbodiimide (540 mg, 2.6 mmol, Aldrich Chemical Company).After 5 minutes, di-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene(240 mg, 770 μmol) was added followed by a catalytic amount of4-dimethylaminopyridine. After 20 min, the reaction mixture was filteredand concentrated (aspirator) to afford 670 mg of oil. The residue wastaken in tetrahydrofuran (4.0 mL) and piperidine (144 mg, 1.7 mmol) wasadded. The reaction was stirred at room temperature for 1 hr and addedto cold diethyl ether. The resulting solid was washed 3× diethyl etherto afford di-(2-methy-4-hydroxymethyl(glycineester)-1,3-dioxolane)-1,4,benzene. Molecular ion calculated forC₂₀H₂₈N₂O₈ 424. found m+1/z 425.2.

Synthesis of Polymers Containing Acid Labile Moieties:

T) Di-(2-methyl-4-hydroxymethyl(glyoxilic acidester)-1,3-dioxolane)-1,4-benzene:1,4-Bis(3-aminopropyl)piperazineCopolymer (1:1) (MC228): To a solution ofdi-(2-methyl-4-hydroxymethyl(glyoxylic acid ester)-1,3-dioxolane)1,4-benzene (100 mg, 0.273 mmol) in dimethylformamide was added1,4-bis(3-aminopropyl)-piperazine (23 μL, 0.273 mmol, Aldrich ChemicalCompany) and the solution was heated to 80° C. After 16 hrs the solutionwas cooled to room temperature and precipitated with diethyl ether. Thesolution was decanted and the residue washed with diethyl ether (2×) anddried under vacuum to afford di-(2-methyl-4-hydroxymethyl(glyoxylic acidester)-1,3-dioxolane) 1,4-benzene: 1,4-bis(3-aminopropyl)-piperazinecopolymer (1:1).

By similar methods, the following polymers were constructed:

-   Di-(2-methyl-4-hydroxymethyl(succinic semialdehyde    ester)-1,3-dioxolane)-1,4-benzene:    -   1,4-Bis(3-aminopropyl)piperazine Copolymer (1:1) (MC208).-   Di-(2-methyl-4-hydroxymethyl(succinic semialdehyde    ester)-1,3-dioxolane)-1,4-benzene:    -   1,4-Bis(3-aminopropyl)piperazine Copolymer (1:1) Reduced with        NaCNBH₃ (MC301).-   Di-(2-methyl-4-hydroxymethyl(succinic semialdehyde    ester)-1,3-dioxolane)-1,4-benzene:    -   1,3-Diaminopropane Copolymer (1:1) (MC300).-   Di-(2-methyl-4-hydroxymethyl(succinic semialdehyde    ester)-1,3-dioxolane)-1,4-benzene:    -   3,3′-Diamino-N-methyldipropylamine Copolymer (1:1) (MC218).-   Di-(2-methyl-4-hydroxymethyl(succinic semialdehyde    ester)-1,3-dioxolane)-1,4-benzene:    -   Tetraethylenepentamine Copolymer (1:1) (MC217).-   Di-(2-methyl-4-hydroxymethyl(glyoxilic acid    ester)-1,3-dioxolane)-1,4-benzene: 1,3-Diaminopropane    -   Copolymer (1:1) (MC226).-   Di-(2-methyl-4-hydroxymethyl(glyoxilic acid    ester)-1,3-dioxolane)-1,4-benzene:    3,3′-Diamino-N-methyldipropylamine Copolymer (1:1) (MC227).

U) Synthesis of 1,4-Bis(3-aminopropyl)piperazine—Glutaric DialdehydeCopolymer (MC140): 1,4-Bis(3-aminopropyl)piperazine (206 μL, 0.998 mmol,Aldrich Chemical Company) was taken up in 5.0 mL H₂O. Glutaricdialdehyde (206 μL, 0.998 mmol, Aldrich Chemical Company) was added andthe solution was stirred at room temperature. After 30 min, anadditional portion of H₂O was added (20 mL), and the mixture neutralizedwith 6 N HCl to pH 7, resulting in a red solution. Dialysis against H₂O(3×3 L, 12,000–14,000 MWCO) and lyophilization afforded 38 mg (14%) ofthe copolymer.

By similar methods, the following polymers were constructed:

-   Diacetylbenzene-1,3-Diaminopropane Copolymer (1:1) (MC321)-   Diacetylbenzene-Diamino-N-methyldipropylamine Copolymer (1:1)    (MC322).-   Diacetylbenzene-1,4-Bis(3-aminopropyl)piperazine Copolymer (1:1)    (MC229)-   Diacetylbenzene-Tetraethylenepentamine Copolymer (1:1) (MC323).-   Glutaric Dialdehyde-1,3-Diaminopropane Copolymer (1:1) (MC324)-   Glutaric Dialdehyde-Diamino-N-methyldipropylamine Copolymer (1:1)    (MC325).-   Glutaric Dialdehyde-Tetraethylenepentamine Copolymer (1:1) (MC326).-   1,4-Cyclohexanone-1,3-Diaminopropane Copolymer (1:1) (MC330)-   1,4-Cyclohexanone-Diamino-N-methyldipropylamine Copolymer (1:1)    (MC33 1).-   1,4-Cyclohexanone-1,4-Bis(3-aminopropyl)piperazine Copolymer (1:1)    (MC312)-   1,4-Cyclohexanone-Tetraethylenepentamine Copolymer (1:1) (MC332).-   2,4-Pentanone-1,4-Bis(3-aminopropyl)piperazine Copolymer (1:1)    (MC340)-   2,4-Pentanone-Tetraethylenepentamine Copolymer (1:1) (MC347).-   1,5-Hexafluoro-2,4-Pentanone-1,4-Bis(3-aminopropyl)piperazine    Copolymer (1:1) (MC339)-   1,5-Hexafluoro-2,4-Pentanone-Tetraethylenepentamine Copolymer (1:1)    (MC346).

V) Synthesis of Poly-L-Glutamic acid (octamer)—Glutaric DialdehydeCopolymer (MC151): H₂N-SEQ ID NO: 11-NHCH₂CH₂NH₂ (; 5.5 mg, 0.0057 mmol,Genosis) was taken up in 0.4 mL H₂O. Glutaric dialdehyde (0.52 μL,0.0057 mmol, Aldrich Chemical Company) was added and the mixture wasstirred at room temperature. After 10 min the solution was heated to 70°C. After 15 hrs, the solution was cooled to room temperature anddialyzed against H₂O (2×2 L, 3500 MWCO). Lyophilization afforded 4.3 mg(73%) poly-glutamic acid (octamer)—glutaric dialdehyde copolymer.

W) Synthesis ofDi-(2-methyl-4-aminomethyl-1,3-dioxolane)-1,4-benzene—GlutaricDialdehyde Copolymer (MC352): To a solution ofdi-(2-methyl-4-aminomethyl-1,3-dioxolane)-1,4-benzene (23 mg, 75 μmol)in dimethylformamide (200 μL) was added glutaric dialdehyde (7.5 mg, 75μmol, Aldrich Chemical Company). The reaction mixture was heated at 80°C. for 6 hrs under nitrogen. The solution was cooled to room temperatureand used without further purification.

X) Synthesis of Di-(2-methy-4-hydroxymethyl(glycineester)-1,3-dioxolane)-1,4,benzene—Glutaric Dialdehyde Copolymer (MC357):To a solution of di-(2-methy-4-hydroxymethyl(glycineester)-1,3-dioxolane)-1,4,benzene (35 mg, 82 μmol) in dimethylformamide(250 μL) was added glutaric dialdehyde (8.2 mg, 82 μmol, AldrichChemical Company). The reaction mixture was heated at 80° C. for 12 hrs.The solution was cooled to room temperature and used without furtherpurification.

Y) Synthesis of Polyvinyl(2-phenyl-4-hydroxymethyl-1,3-dioxolane) fromthe reaction of Polyvinylphenyl Ketone and Glycerol: Polyvinyl phenylketone (500 mg, 3.78 mmol, Aldrich Chemical Company) was taken up in 20mL dichloromethane. Glycerol (304 μL, 4.16 mmol, Acros Chemical Company)was added followed by p-toluenesulfonic acid monohydrate (108 mg, 0.57mmol, Aldrich Chemical Company). Dioxane (10 mL) was added and thesolution was stirred at room temperature overnight. After 16 hrs, TLCindicated the presence of ketone. The solution was concentrated underreduced pressure, and the residue dissolved in dimethylformamide (7 mL).The solution was heated to 60° C. for 16 hrs. After 16 hrs, TLCindicated the ketone had been consumed. Dialysis against H₂O (1×3 L,3500 MWCO), followed by lyophilization resulted in 606 mg (78%) of theketal. Ketone was not observed in the sample by TLC analysis, however,upon treatment with acid, the ketone was again detected.

Z) Synthesis of Polyvinyl(2-methyl-4-hydroxymethyl(succinic anhydrideester)-1,3-dioxolane: To a solution ofpolyvinyl(2-methyl-4-hydroxymethyl-1,3-dioxolane) (220 mg, 1.07 mmol) indichloromethane (5 mL) was added succinic anhydride (161 mg, 1.6 mmol,Sigma Chemical Company), followed by diisopropylethyl amine (0.37 mL,2.1 mmol, Aldrich Chemical Company) and the solution was heated atreflux. After 16 hrs, the solution was concentrated, dialyzed againstH₂O (1×3 L, 3500 MWCO), and lyophilized to afford 250 mg (75%) of theketal acid polyvinyl(2-methyl-4-hydroxymethyl(succinic anhydrideester)-1,3-dioxolane.

AA) Synthesis of Ketal from Polyvinyl Alcohol and 4-Acetylbutyric Acid:Polyvinylalcohol (200 mg, 4.54 mmol, 30,000–60,000 MW, Aldrich ChemicalCompany) was taken up in dioxane (10 mL). 4-acetylbutyric acid (271 μL,2.27 mmol, Aldrich Chemical Company) was added followed byp-toluenesulfonic acid monohydrate (86 mg, 0.45 mmol, Aldrich ChemicalCompany). After 16 hrs, TLC indicated the presence of ketone. Thesolution was concentrated under reduced pressure, and the residuedissolved in dimethylformamide (7 mL). The solution was heated to 60° C.for 16 hrs. After 16 hrs, TLC indicated the loss of ketone in thereaction mixture. Dialysis against H₂O (1×4 L, 3500 MWCO), followed bylyophilization resulted in 145 mg (32%) of the ketal. Ketone was notobserved in the sample by TLC analysis, however, upon treatment withacid, the ketone was again detected.

AB) Partial Esterification of Poly-Glutamic Acid withDi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (MC 196): To asolution of poly-L-glutamic acid (103 mg, 792 μmol, 32,000 MW, SigmaChemical Company) in sodium phosphate buffer (30 mM) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (129 mg, 673μmol, Aldrich Chemical Company), followed bydi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (25.0 mg, 80.5μmol), and a catalytic amount of 4-dimethylaminopyridine. After 12 hrs,the reaction mixture was dialyzed against water (2×1 L, 12,000–14,000MWCO) and lyophilized to afford 32 mg of poly-glutamic acid partiallyesterified with di-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene.

AC) Aldehyde Derivatization of the Poly-Glutamic Acid PartiallyEsterified with Di-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene:To a solution of succinic semialdehyde (2.4 mg, 23 μmol, AldrichChemical Company) in water (100 μL) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.7 mg, 2.4μmol, Aldrich Chemical Company) followed by N-hydroxysuccinimide (2.8mg, 24 μmol, Aldrich Chemical Company). The reaction was stirred at roomtemperature for 20 min. Formation of the N-hydroxysuccinic ester ofsuccinic semialdehyde was confirmed by mass spectrometry.

Poly-glutamic acid partially esterified withdi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (15.0 mg, 115μmol) was taken up in water (100 μL) and added to the N-hydroxysuccinicester of succinic semialdehyde, followed by a crystal of4-dimethylaminopyridine. The reaction mixture was stirred overnight atroom temperature. After 12 hrs the reaction mixture was dialyzed againstwater (2×1 L, 12,000–14,000 MWCO) and lyophilized to afford 3.0 mg.After dialysis the product tested positive for aldehyde content with2,4-di-nitrophenylhydrazine.

AD) Synthesis of a Silyl Ether from Polyvinylalcohol and3-Aminopropyl-trimethoxysilane (MC221): To a solution ofpolyvinylalcohol (520 mg, 11.8 mmol (OH), 30,000–70,000 MW, SigmaChemical Company) in dimethylformamide (4 mL) was added3-aminopropyltrimethoxysilane (1.03 mL, 5.9 mmol, Aldrich ChemicalCompany) and the solution was stirred at room temperature. After 2.5hrs, a 20 μL aliquot of the reaction mixture was removed and added topDNA (pCI Luc) (100 μg) in 25 mM HEPES buffer at pH 7.5 (500 μL) to testfor polyamine formation (pDNA:amine 1:3). Particle sizing (BrookhavenInstruments Coporation, ZetaPlus Particle Sizer, I90, 532 nm) indicatedan effective diameter of 3000 nm (1.3 mcps) indicating pDNA condensationand particle formation. An aliquot of 1 N HCl (40 μL) was added to thesample, and the particle size was again measured. After 1 min ofexposure to the acidic conditions, the particle size was 67,000 nm (600kcps). After 10 min, particles were no longer present within the sample.The sample was dried under high vacuum to afford 1.0 g (83%) whitesolid.

By similar methods, the following polymers were constructed:

-   Silyl Ether from Poly-L-Arginine/-L-Serine(3:1) and    3-Aminopropyltrimethoxysilane (2:1) (MC358):    Poly-L-Arginine/-L-Serine(3:1) (20,000–50,000 MW, Sigma Chemical    Company), 3-Aminopropyltrimethoxysilane (Aldrich Chemical Company)-   Silyl Ether from Poly-DL-Serine and 3-Aminopropyltrimethoxysilane    (3:1) (MC366): Poly-DL-Serine (5,000–15,000 MW, Sigma Chemical    Company), 3-Aminopropyl-trimethoxysilane (Aldrich Chemical Company)-   Silyl Ether from Poly-DL-Serine and 3-Aminopropyltrimethoxysilane    (2:1) (MC367). Poly-DL-Serine (5,000–15,000 MW, Sigma Chemical    Company), 3-Aminopropyl-trimethoxysilane (Aldrich Chemical Company)-   Silyl Ether from Poly-DL-Serine and    N-[3-(Triethoxysilyl)propyl]-4,5-dihydroimidizole (3:1) (MC369):    Poly-DL-Serine (5,000–15,000 MW, Sigma Chemical Company)-   N-[3-(Triethoxysilyl)propyl]-4,5-dihydroimidizole (United Chemical    Technologies, Incorporated)-   Silyl Ether from Poly-DL-Serine and    N-Trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (3:1)    (MC370): Poly-DL-Serine (5,000–15,000 MW, Sigma Chemical Company)-   N-Trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (United    Chemical Technologies, Incorporated)-   Silazane from Poly-L-Lysine and 3-Aminopropyltrimethoxysilane (2:1)    (MC360). Poly(1,1-Dimethylsilazane) Tolemer (MC222): Sample was    obtained from United Chemical Technologies, Incorporated.

Example 2

Transfection with pH-Sensitive Compounds and/or Membrane Active Agents

A) In Vitro Transfection with DNA-PLL-KL₃ and dimethylmaleamic KL₃: To acomplex of plasmid DNA pCIluc (10 μg/mL, 0.075 mM in phosphate, 2.6μg/μL pCIluc; prepared according to Danko, I., Williams, P., Herweijer,H. Zhang, G., Latendresse, J. S., Bock, I., Wolff, J. A. Hum. Mol.Genetics 1997, 6, 1435.) and poly-L-lysine (40 μg/mL) in 0.5 mL of 5 mMHEPES pH 7.5 was added succinylated poly-L-lysine (34,000 MW, AldrichChemical), 2,3-dimethylmaleamic melittin and 2,3-dimethylmaleamic KL₃.The DNA-poly-L-lysine-2,3-dimethylmaleamic peptide complexes were thenadded (200 μL) to wells containing 3T3 mouse embryonic fibroblast cellsin 290 mM glucose and 5 mM HEPES buffer pH 7.5. After 1.5 h, the glucosemedia was replaced with Dubelco's modified Eagle Media and the cellswere allowed to incubate for 48 h. The cells were then harvested andthen assayed for luciferase expression as previously reported (Wolff, J.A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used. The amount of transfection was reported inrelative light units and is the average transfection for two separatewells of cells.

Relative Light Units (Relative to Peptide Succinylated poly-L-lysine)Succinylated poly-L-lysine 6410 (1) KL₃ 261 (0.04) 2,3-dimethylmaleamicKL₃ 49535 (7.7)

B) In Vitro Transfection with DNA-PLL complexes with dimethylmaleamicKL₃ and dimethylmaleamic KL₃-PLL: To a complex of plasmid DNA pCIluc (10μg/mL, prepared according to Danko, I., Williams, P., Herweijer, H.Zhang, G., Latendresse, J. S., Bock, I., Wolff, J. A. Hum. Mol. Genetics1997, 6, 1435.) and poly-L-lysine (40 μg/mL) in 0.5 mL water was added10 mg of 2,3-dimethylmaleamic -KL₃-PLL or 2,3-dimethylmaleamic -KL₃. TheDNA-poly-L-lysine-2,3-dimethylmaleamic peptide complexes were then added(200 μL) to a well containing 3T3 mouse embryonic fibroblast cells inopti-MEM. After 4 h, the media was replaced with 90% Dubelco's modifiedEagle Media and 10% fetal bovine serum the cells were then allowed toincubate for 48 h. The cells were then harvested and then assayed forluciferase expression as previously reported (Wolff, J. A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.Direct gene transfer into mouse muscle in vivo. Science, 1465–1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used. The amount of transfection was reported inrelative light units and is the average transfection for two separatewells of cells.

2,3-dimethylmaleamic peptide Relative Light Units 2,3-dimethylmaleamicKL₃  20927 2,3-dimethylmaleamic KL₃-PLL 130478

C) In Vitro Transfection with DNA-PLL complexes with dimethylmaleamicKL₃-PLL, 2-propionic-3-methylmaleamic KL₃-PLL, and succinimic KL₃-PLL:To a complex of plasmid DNA pCIluc (10 μg/mL, prepared according toDanko, I., Williams, P., Herweijer, H. Zhang, G., Latendresse, J. S.,Bock, I., Wolff, J. A. Hum. Mol. Genetics 1997, 6, 1435.) andpoly-L-lysine (40 μg/mL) in 0.5 mL water was added 25 μg of2,3-dimethylmaleamic -KL₃-PLL, 2-propionic-3-methylmaleamic KL₃-PLL, andsuccinimic KL₃-PLL. The DNA-poly-L-lysine-peptide complexes were thenadded (200 μL) to a well containing 3T3 mouse embryonic fibroblast cellsin opti-MEM media. After 4 h, the media was replaced with 90% Dubelco'smodified Eagle Media and 10% fetal bovine serum the cells were thenallowed to incubate for 48 h. The cells were then harvested and thenassayed for luciferase expression as previously reported (Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used. The amount of transfection was reported inrelative light units and is the average transfection for two separatewells of cells.

Modified peptide Relative Light Units 2,3-dimethylmaleamic KL₃-PLL 96221 2-propionic-3-methylmaleamic KL₃-PLL 102002 succinimic KL₃-PLL 21206

D) In Vitro Transfection with DNA-PLL with 2,3-dimethylmaleamic-modifiedlipids: To a complex of plasmid DNA pCIluc (10 μg/mL, 2.2 μg/μL pCIluc;prepared according to Danko, I., Williams, P., Herweijer, H. Zhang, G.,Latendresse, J. S., Bock, I., Wolff, J. A. Hum. Mol. Genetics 1997, 6,1435.) and poly-L-lysine (40 μg/mL) in 0.5 mL of deionized water wasadded 800 μg glycine followed by 40 μg 2,3-dimethylmaleic DOG,2,3-dimethylmaleamicMC213, 2,3-dimethylmaleamicMC303, or2,3-dimethylmaleamic-DOPE. TheDNA-poly-L-lysine-2,3-dimethylmaleamic-modified lipids were then added(200 μL) to a well containing opti-MEM media. After 4 h, the media wasreplaced with 90% Dubelco's modified Eagle Media and 10% fetal bovineserum the cells were then allowed to incubate for 48 h. The cells werethen harvested and then assayed for luciferase expression as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection fortwo separate wells of cells.

Relative Light Units 2,3-dimethylmaleamic-modified lipids (Relativetopoly-L-lysine) No lipid 6000 (1) MC213 91356 (15) MC303 469756 (78)DOPE 243359 (40) DOG 193624 (32)

E) Transfection of HELA Cells with Histone H1 and the Membrane Activepeptide melittin, dimethylmaleic modified melittin or succinic anhydridemodifed melittin:

Three Complexes were Formed:

-   Complex I) To 300 μL Opti-MEM was added Histone H1(12 μg, Sigma    Corporation) followed by the peptide Melittin (20 μg) followed by    pDNA (pCI Luc, 4 μg).-   Complex II) To 300 μL Opti-MEM was added Histone H1(12 μg, Sigma    Corporation) followed by the 2,3-dimethylmaleic modified peptide    Melittin (20 μg) followed by pDNA (pCI Luc, 4 μg).-   Complex III) To 300 μL Opti-MEM was added Histone H1(12 μg, Sigma    Corporation) followed by the succinic anhydride modified peptide    Melittin (20 μg) followed by pDNA (pCI Luc, 4 μg).

Transfections were carried out in 35 mm wells. At the time oftransfection, HELA cells, at approximately 60% confluency, stored incomplete growth media, DMEM with 10% fetal bovine serum (Sigma). 150 μLof complex was added to each After an incubation of 48 hours, the cellswere harvested and the lysate was assayed for luciferase expression aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer intomouse muscle in vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used. The amount oftransfection was reported in relative light units and is the averagetransfection for two separate wells of cells.

Results:

-   Complex I: RLU=2,161-   Complex II: RLU=105,909-   Complex III: RLU=1,056

The 2,3-dimethylmaleic modification of the peptide melittin allows thepeptide to complex with the cationic protein Histone H1 and then cleaveto release and reactivate in the lowered pH encountered by the complexin the cellular endosomal compartment. This caused a significantincrease in luciferase expression over either unmodified melittinpeptide or melittin peptide modified with succinic anhydride whichallows complexing with Histone H1 but will not cleave in lowered pH.

F) Transfection of 3T3 Cells with Dioleoyl1,2-Diacyl-3-Trimethylammonium-Propane (DOTAP) and the membrane activepeptide KL3 conjugated to dimethylmaleic modified Polyallylamine(DM-PAA-KL3) and poly-L-lysine orL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer.

Three Complexes were Formed:

-   Complex I) To 250 μL 25 mM HEPES pH8.0 was added DOTAP 300 μg,    Avanti Polar Lipids Inc)-   Complex II) To 250 μL 25 mM HEPES pH8.0 was added DOTAP(300 μg,    Avanti Polar Lipids Inc) followed by DM-PAA-KL₃ (10 μg) followed by    poly-L-lysine (10 μg, Sigma).-   Complex III) To 250 μL 25 mM HEPES pH8.0 was added DOTAP(300 μg,    Avanti Polar Lipids Inc) followed by DM-PAA-KL₃ (10 μg) followed by    L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer (10 μg).

Liposomes for each complex were formed by 5 minutes of bath sonicationthen purified in batch by addition of 250 ul of DEAE sephadex A-25. DNA(25 ug, pCILuc)was then added to the supernatant containing the purifiedliposomes of each complex.

Transfections were carried out in 35 mm wells. At the time oftransfection, 3T3 cells,at approximately 60% confluency, stored incomplete growth media, DMEM with 10% fetal bovine serum (Sigma). 50 μLof complex was added to each well. After an incubation of 48 hours, thecells were harvested and the lysate was assayed for luciferaseexpression as previously reported (Wolff, J. A., Malone, R. W.,Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. Directgene transfer into mouse muscle in vivo. Science, 1465–1468, 1990.). ALumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer wasused. The amount of transfection was reported in relative light unitsand is the average transfection for two separate wells of cells.

Results:

-   Complex I: RLU=167-   Complex II: RLU=60,092-   Complex III: RLU=243,986

The 2,3-dimethylmaleic modification of DM-PAA-KL3 allows the polymer tocomplex with the cationic polymerL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer and then cleavageof the 2,3-dimethylmaleamic group to release and reactivate in thedisulfide reducing environment encountered by the complex in the cell.This caused a significant increase in luciferase expression over eitherDOTAP complexes alone or DM-PAA-KL3 complexed with poly-L-lysine thatwill not cleave in the reducing environment encountered by the complexin the cell.

G) Transfection of3T3 Cells with Complexes of pCI Luc pDNA/CationicPolymers Caged with Compounds Containing Acid Labile Moieties.

Several Complexes were Formed:

-   Complex I: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added LT-1® (60 μg, Mirus Corporation).-   Complex II: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added PLL (36 μg in 3.6 μL H₂O, 32,000 MW, Sigma Chemical    Company).-   Complex III: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added PLL (36 μg in 3.6 μL H₂O, 32,000 MW, Sigma Chemical    Company) followed by DTBP (60 μg in 6 μL H₂O, Pierce Chemical    Company).-   Complex IV: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added PLL (36 μg in 3.6 μL H₂O, 32,000 MW, Sigma Chemical    Company) followed by DTBP (60 μg in 6 μL H₂O, Pierce Chemical    Company) followed by N,N′-dioleoyl-1,4-bis(3-aminopropyl)piperazine    (10 μg, 2 μg/μL in EtOH).-   Complex V: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added PLL (36 μg in 3.6 μL H₂O, 32,000 MW, Sigma Chemical    Company) followed by MC211 (87 μg in 8.7 μL dimethylformamide).-   Complex VI: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added PLL (36 μg in 3.6 μL H₂O, 32,000 MW, Sigma Chemical    Company) followed by MC211 (87 μg in 8.7 μL dimethylformamide)    followed by N,N′-dioleoyl-1,4-bis(3-aminopropyl)piperazine (10 μg, 2    μg/μL in EtOH).-   Complex VII: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added Histone H1 (120 μg in 12 μL H₂O, Sigma Chemical Company).-   Complex VIII: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added Histone H1 (120 μg in 12 μL H₂O, Sigma Chemical Company)    followed by DTBP (100 μg in 10 μL H₂O, Pierce Chemical Company).-   Complex IX: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added Histone H1 (120 μg in 12 μL H₂O, Sigma Chemical Company)    followed by DTBP (100 μg in 10 μL H₂O, Pierce Chemical Company)    followed by N,N′-dioleoyl-1,4-bis(3-aminopropyl)piperazine (10 μg, 2    μg/μL in EtOH).-   Complex X: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added Histone H1 (120 μg in 12 μL H₂O, Sigma Chemical Company)    followed by MC211 (145 μg in 14.5 μL dimethylformamide).-   Complex XI: To a solution of pDNA (pCI Luc, 20 μg) in H₂O (400 μL)    was added Histone H1 (120 μg in 12 μL H₂O, Sigma Chemical Company)    followed by MC211 (145 μg in 14.5 μL dimethylformamide) followed by    N,N′-dioleoyl-1,4-bis(3-aminopropyl)piperazine (10 μg, 2 μg/μL in    EtOH).    Transfections were carried out in 35 mm wells. At the time of    transfection, 3T3 cells, at approximately 50% confluency, were    washed once with PBS (phosphate buffered saline), and subsequently    stored in serum-free media (2.0 mL, Opti-MEM, Gibco-BRL). 100 μL of    complex was added to each well. After a 3.25 h incubation period at    37° C., the media containing the complexes was aspirated from the    cells, and replaced with complete growth media, DMEM with 10% fetal    bovine serum (Sigma). After an additional incubation of 48 hours,    the cells were harvested and the lysate was assayed for luciferase    expression as previously reported (Wolff, J. A., Malone, R. W.,    Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.    Direct gene transfer into mouse muscle in vivo. Science, 1465–1468,    1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)    luminometer was used. The amount of transfection was reported in    relative light units and is the average transfection for two    separate wells of cells.

Results: Complex I: 2,467,529 RLU Complex II: 10,748 RLU Complex III:377 RLU Complex IV: 273 RLU Complex V: 7,174 RLU Complex VI: 71,338 RLUComplex VII: 162,166 RLU Complex VIII: 1,336 RLU Complex IX: 162,166 RLUComplex X: 51,003 RLU Complex XI: 3,949,177 RLUThe transfection results indicate that caging cationic pDNA complexes(PLL or Histone H1) with DTBP reduce the amount of expressed luciferine.Caging of the cationic pDNA complexes with MC211 results in an increasedamount of expressed luciferine relative to the DTBP examples.In Vivo Transfections

H) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/PolymerContaining Acid Labile Moieties:

H1) Mouse Tail Vein Injections of Complexes of pDNA (pCILuc)/1,4-Bis(3-aminopropyl)piperazine Glutaric Dialdehyde Copolymer (MC140).

Three Complexes were Prepared as Follows:

-   Complex I: pDNA (pCI Luc, 50 μg) in 12.5 mL Ringers.-   Complex II: pDNA (pCI Luc, 50 μg) was mixed with    1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer (50    μg) in 1.25 mL HEPES 25 mM, pH 8. This solution was then added to    11.25 mL Ringers.-   Complex III: pDNA (pCI Luc, 50 μg) was mixed with poly-L-lysine    (94.5 μg, MW 42,000, Sigma Chemical Company) in 12.5 mL Ringers.    2.5 mL tail vein injections of 2.5 mL of the complex were preformed    as previously described. Luciferase expression was determined as    previously reported (Wolff, J. A., Malone, R. W., Williams, P.,    Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene    transfer into mouse muscle in vivo. Science, 1465–1468, 1990.). A    Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer was    used.

Results: 2.5 mL injections Complex I: 3,692,000 RLU Complex II:1,047,000 RLU Complex III: 4,379 RLUResults indicate an increased level of pCI Luc DNA expression inpDNA/1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymercomplexes over pCI Luc DNA/poly-L-lysine complexes. These results alsoindicate that the pDNA is being released from thepDNA/1,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde copolymercomplexes, and is accessible for transcription.

H2A) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ImineContaining Copolymer's: By similar methods described above, severaladditional complexes were prepaired from imine containing polymers at a3:1 charge ratio of polycation to pDNA.

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50 μg)/MC2292.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I: n = 3 3,430,000 RLU Complex II: n= 3 21,400,000 RLUThe results indicate that the pDNA is being released from the pDNA/iminecontaining copolymer complexes, and is accessible for transcription.

H2B) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ImineContaining Copolymer's. By similar methods described above, severaladditional complexes were prepaired from imine containing polymers at a3:1 charge ratio of polycation to pDNA.

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50μg)/MC140-2 Complex III: pDNA (pCI Luc, 50 μg)/MC3122.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I: n = 1 9,460,000 RLU Complex II: n= 3 7,730,000 RLU Complex III: n = 3 16,300,000 RLUThe results indicate that the pDNA is being released from the pDNA/iminecontaining copolymer complexes, and is accessible for transcription.

H3A) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/KetalContaining Copolymers. By similar methods described above, severalcomplexes were prepared at a 3:1 charge ratio of polycation to pDNA:

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50μg)/PLL-DTBP(Pierce Chemical Co., 50%) Complex III: pDNA (pCI Luc, 50μg)/PLL-MC211(50%) Complex IV: pDNA (pCI Luc, 50 μg)/MC228 Complex V:pDNA (pCI Luc, 50 μg)/MC2082.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I: n = 3 2,440,000 RLU Complex II: n= 3 110,000 RLU Complex III: n = 3 292,000 RLU Complex IV: n = 3 119,000RLU Complex V: n = 3 3,590,000 RLU

H3B) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/KetalContaining Copolymers. By similar methods described above, severalcomplexes were prepared at a 3:1 charge ratio of polycation to pDNA:

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50μg)/PLL-MC225(50%) Complex III: pDNA (pCI Luc, 50 μg)/MC217 Complex IV:pDNA (pCI Luc, 50 μg)/MC2182.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I: n = 3 5,940,000 RLU Complex II: n= 3 611,000 RLU Complex III: n = 3 5,220,000 RLU Complex IV: n = 37,570,000 RLU

H3C) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/KetalContaining Copolymers. By similar methods described above, severalcomplexes were prepared at a 3:1 charge ratio of polycation to pDNA:

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50 μg)/MC208Complex III: pDNA (pCI Luc, 50 μg)/MC3012.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I: n = 3 3,430,000 RLU Complex II: n= 2 9,110,000 RLU Complex III: n = 3 8,570,000 RLUResults indicate an increased level of pCI Luc DNA expression in ComplexIII and Complex VII relative to Complex II indicating that when the acidlabile homobifunctional amine reactive system (MC211, MC225) is used,more pDNA is accessible for transcription relative to the non-labilehomobifunctional amine reactive system (DTBP). These results alsoindicate that the pDNA is being released from the pDNA/ketal containingcopolymer complexes, and is accessible for transcription.

H4) Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/SiliconContaining Polymers. By similar methods described above, severalcomplexes were prepared at a 3:1 charge ratio of polycation to pDNA:

Complex I: pDNA (pCI Luc, 50 μg) Complex II: pDNA (pCI Luc, 50 μg)/MC221Complex III: pDNA (pCI Luc, 50 μg)/MC222 Complex IV: pDNA (pCI Luc, 50μg)/MC223 Complex V: pDNA (pCI Luc, 50 μg)/MC358 Complex VI: pDNA (pCILuc, 50 μg)/MC358 recharged with SPLL (MC359) Complex VII: pDNA (pCILuc, 50 μg)/MC360 Complex VIII: pDNA (pCI Luc, 50μg)/Poly-L-Arginine/-L-Serine(3:1) Complex IX: pDNA (pCI Luc, 50μg)/MC366 Complex X: pDNA (pCI Luc, 50 μg)/MC367 Complex XI: pDNA (pCILuc, 50 μg)/MC369 Complex XII: pDNA (pCI Luc, 50 μg)/MC3702.5 mL tail vein injections of 2.5 mL of the complex were preformed aspreviously described. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used. The amount of transfectionwas reported in relative light units and is the average transfection forn separate animals.

Results: 2.5 mL injections Complex I:  n = 14 14,564,000 RLU Complex II: n = 14 14,264,000 RLU Complex III: n = 9 13,449,000 RLU Complex IV: n =3 6,927,000 RLU Complex V: n = 3 10,049,000 RLU Complex VI: n = 313,879,000 RLU Complex VII: n = 3 10,599,000 RLU Complex VIII: n = 3638,000 RLU Complex IX: n = 3 12,597,000 RLU Complex X: n = 3 13,093,000RLU Complex XI: n = 3 25,129,000 RLU Complex XII: n = 3 15,857,000 RLUThe results indicate that the pDNA is being released from thepDNA/Silicon containing polycation complexes, and is accessible fortranscription. Additionally, the results indicate that complex VIII(does not contain the silicon) is much less effective in the assay thanis complex V. Additionally, the results indicate that upon the additionof a third layer, a polyanion (complex VI), the complex containing thesilicon polymer allows for pDNA transcription.

G) Mouse Intramuscular Injections of Complexes of pDNA (pCI Luc)/PolymerContaining Acid Labile Moiety(s): Complexes were prepared as follows:

Complex I: pDNA. pDNA (pCI Luc, 60 μg, 27 μl) was added to 0.9% saline(1173 μL). Complex II: pDNA/MC208 (1:0.5). To a solution of pDNA (pCILuc, 60 μg, 27 μL) in 0.9% saline (1173 μL) was added MC208 (0.19 μL, indimethylformamide). Complex III: pDNA/MC208 (1:3). To a solution of pDNA(pCI Luc, 60 μg) in 0.9% saline (1161 μL) was added MC208 (12 μL, indimethylformamide). Complex IV: pDNA/MC301 (1:0.5). To a solution ofpDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline (1173 μL) was added MC301(0.15 μL, in dimethylformamide). Complex V: pDNA/MC301 (1:3). To asolution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline (1172 μL) wasadded MC301 (0.88 μL, in dimethylformamide). Complex VI: pDNA/MC229(1:0.5). To a solution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline(1173 μL) was added MC229 (0.09 μL, in dimethylformamide). Complex VII:pDNA/MC229 (1:3). To a solution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9%saline (1172 μL) was added MC229 (0.59 μL, in dimethylformamide).Complex VIII: pDNA/MC140 (1:0.5). To a solution of pDNA (pCI Luc, 60 μg,27 μL) in 0.9% saline (1173 μL) was added MC140 (0.08 μL, indimethylformamide). Complex IX: pDNA/MC140 (1:3). To a solution of pDNA(pCI Luc, 60 μg, 27 μL) in 0.9% saline (1173 μL) was added MC140 (0.48μL, in dimethylformamide). Complex X: pDNA/MC312 (1:0.5). To a solutionof pDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline (1173 μL) was added MC312(0.08 μL, in dimethylformamide). Complex XI: pDNA/MC312 (1:3). To asolution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline (1173 μL) wasadded MC312 (0.50 μL, in dimethylformamide). Complex XII: pDNA/MC217(1:0.5). To a solution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9% saline(1173 μL) was added MC217 (0.11 μL, in dimethylformamide). Complex XIII:pDNA/MC217 (1:3). To a solution of pDNA (pCI Luc, 60 μg, 27 μL) in 0.9%saline (1172 μL) was added MC217 (0.69 μL, in dimethylformamide).Complex XIV: pDNA/MC221 (1:3). To a solution of pDNA (pCI Luc, 40 μg, 18μL) in 0.9% saline (781 μL) was added MC221 (1.1 μL, in H₂O). ComplexXV: pDNA/MC222 (1:3). To a solution of pDNA (pCI Luc, 40 μg, 18 μL) in0.9% saline (782 μL) was added MC222 (0.40 μL, in H₂O). Complex XVI:pDNA. pDNA (pCI Luc, 100 μg, 45 μL) was added to 0.9% saline (1955 μL).Complex XVII: pDNA/PLL (1:3). To a solution of pDNA (pCI Luc, 100 μg, 45μL) in 0.9% saline (1943 μL) was added PLL (32,000 MW, Sigma ChemicalCompany, 12 μL, in H₂O). Complex XVIII: pDNA/PEI (1:3). To a solution ofpDNA (pCI Luc, 100 μg, 45 μL) in 0.9% saline (1,945 μL) was added PEI(25,000 MW, Sigma Chemical Company, 10 μL (10 mg/mL), in H₂O). ComplexXIX: pDNA/HistoneH1 (1:3). To a solution of pDNA (pCI Luc, 100 μg, 45μL) in 0.9% saline (1.889 μL) was added Histone H1 (Sigma ChemicalCompany, 66 μL (10 mg/mL), in H₂O).Direct muscle injections of 200 μL of the complex were preformed aspreviously described (See Budker, V., Zhang, G., Danko, I., Williams,P., and Wolff, J., “The Efficient Expression Of IntravascularlyDelivered DNA In Rat Muscle,” Gene Therapy 5, 272–6(1998); Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465–1468, 1990. Seven days post injection, the animals were sacrificed,and the muscle harvested. Samples were homogenized in lux buffer (1 mL),and centrifuged for 15 minutes at 4000 RPM. Luciferase expression wasdetermined as previously reported. Results reported are for the averageexpression for the quadracep muscle (left and right quadracep muscle/2)per number of animals (n).

Results: Complex I: n = 3 473,148 RLU Complex II: n = 3 328,054 RLUComplex III: n = 3 104,348 RLU Complex IV: n = 3 228,582 RLU Complex V:n = 3 259,007 RLU Complex VI: n = 3 989,905 RLU Complex VII: n = 3286,118 RLU Complex VIII: n = 3 433,177 RLU Complex IX: n = 3 46,727 RLUComplex X: n = 3 365,440 RLU Complex XI: n = 3 454 RLU Complex XII: n =3 1,386,208 RLU Complex XIII: n = 3 295 RLU Complex XIV: n = 2 352,639RLU Complex XV: n = 2 459,695 RLU Complex XVI:  n = 10 1,281,401 RLUComplex XVII:  n = 10 2,789 RLU Complex XVIII:  n = 10 340 RLU ComplexXIX:  n = 10 357 RLUThe complexes prepared from pCI Luc DNA and polymers containing acidlabile moities are effective in direct muscle injections. The luciferaseexpression indicates that the pDNA is being released from the complexand is accessible for transcription.

Example 3

Synthesis of Peptides and Polyions

A) Peptide synthesis. Peptide syntheses were performed using standardsolid phase peptide techniques using FMOC chemistry. N-terminal acryloyl6-aminohexanoyl-SEQ ID NO: 10-CO₂ (AcKL₃) was synthesized according topublished procedure (O'Brien-Simpson, N. M., Ede, N. J., Brown, L. E.,Swan, J., Jackson, D. C J. Am. Chem. Soc. 1997, 119, 1183).

B) Coupling KL₃ to poly(allylamine). To a solution of poly(allylamine)(2 mg) in water (0.2 mL) was added KL3 (0.2 mg, 2.5 eq) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1 mg, 150eq). The reaction was allowed to react for 16 h and then the mixture wasplaced into dialysis tubing and dialyzed against 3×1 L for 48 h. Thesolution was then concentrated by lyophilization to 0.2 mL.

C) Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. Toa solution of N,N′-Bis(t-BOC)-L-cystine (85 mg, 0.15 mmol) in ethylacetate (20 mL) was added N,N′-dicyclohexylcarbodiimide (108 mg, 0.5mmol) and N-hydroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, thesolution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (54 mL, 0.25 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h. The ethylacetate was then removed by rotary evaporation and the resulting solidwas dissolved in trifluoroacetic acid (9.5 mL), water (0.5 mL) andtriisopropylsilane (0.5 mL). After 2 h, the trifluoroacetic acid wasremoved by rotary evaporation and the aqueous solution was dialyzed in a15,000 MW cutoff tubing against water (2×2 l) for 24 h. The solution wasthen removed from dialysis tubing, filtered through 5 μM nylon syringefilter and then dried by lyophilization to yield 30 mg of polymer.

D) Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer.1,4-Bis(3-aminopropyl)piperazine (10 mL, 0.050 mmol, Aldrich ChemicalCompany) was taken up in 1.0 mL methanol and HCl (2 mL, 1 M in Et2O,Aldrich Chemical Company) was added. Et2O was added and the resultingHCl salt was collected by filtration. The salt was taken up in 1 mL DMFand 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (30 mg, 0.050 mmol)was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (35 mL, 0.20 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr. the solution was cooled, diluted with 3 mLH2O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 23 mg (82%) of5,5′-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)piperazinecopolymer.

E) Synthesis of polypropylacrylic acid. To a solution ofdiethylpropylmalonate (2 g, 10 mmol) in 50 mL ethanol was addedpotassium hydroxide (0.55 g, 1 eq) and the mixture was stirred at roomtemperature for 16 hours. The ethanol was then removed by rotaryevaporation. The reaction mixture was partitioned between 50 mL ethylacetate and 50 mL of water. The aqueous solution was isolated, andacidified with hydrochloric acid. The solution was again partitionedbetween ethyl acetate and water. The ethyl acetate layer was isolated,dried with sodium sulfate, and concentrated to yield a clear oil. Tothis oil was added 20 mL of pyridine, paraformaldehyde (0.3 g, 10 mmol),and 1 mL piperidine. The mixture was refluxed at 130° C. until theevolution of gas was observed, ca. 2 hours. The ester product was thendissolved into 100 mL ethyl ether, which was washed with 100 mL 1Mhydrochloric acid, 100 mL water, and 100 mL saturated sodiumbicarbonate. The ether layer was isolated, dried with magnesium sulfate,and concentrated by rotary evaporation to yield a yellow oil. The esterwas then hydrolyzed by dissolving in 50 mL ethanol with addition ofpotassium hydroxide (0.55 gm, 10 mmol). After 16 hours, the reactionmixture was acidified by the addition of hydrochloric acid. Thepropylacrylic acid was purified by vacuum distillation (0.9 g, 80%yield), boiling point of product is 60° C. at 1 torr. The propylacrylicacid was polymerized by addition of 1 mole percent ofazobisisobutyonitrile and heating to 60° C. for 16 hours. Thepolypropylacrylic acid was isolated by precipitation with ethyl ether.

F) Synthesis of poly N-terminal acryloyl 6-aminohexanoyl-SEQ ID NO:10-CO₂ (pAcKL₃). A solution of AcKL3 (20 mg, 7.7 μmol) in 0.5 mL of 6 Mguanidinium hydrochloride, 2 mM EDTA, and 0.5 M Tris pH 8.3 was degassedby placing under a 2 torr vacuum for 5 minutes. Polymerization of theacrylamide was initiated by the addition of ammonium persulfate (35 μg,0.02 eq.) and N,N,N,N-tetramethylethylenediamine (1 μL). Thepolymerization was allowed to proceed overnight. The solution was thenplaced into dialysis tubing (12,000 molecular weight cutoff) anddialyzed against 3×2 L over 48 hours. The amount of polymerized peptide(6 mg, 30% yield) was determined by measuring the absorbance of thetryptophan residue at 280 nm, using an extinction coefficient of 5690cm⁻¹M⁻¹ (Gill, S. C. and von Hippel, P. H. Analytical Biochemistry(1989) 182, 319–326)

Example 4

Kinetic Analysis

A) Kinetics of conversion of dimethyl maleamic modified poly-L-lysine topoly-L-lysine. Dimethyl maleamic modified poly-L-lysine (10 mg/mL) wasincubated in 10 mM sodium acetate buffer pH 5. At various times,aliquots (10 μg) were removed and added to 0.5 mL of 100 mM boraxsolution containing 0.4 mM trinitrobenzenesulfonate. A half an hourlater, the absorbance of the solution at 420 nm was measured. Todetermine the concentration of amines at each time point, the extinctioncoefficient was determine for the product of trinitrobenzenesulfonateand poly-L-lysine. Using this extinction coefficient we were able tocalculate the amount of amines and maleamic groups at each time point. Aplot of ln (A_(t)/A₀) as a function of time was a straight line whoseslope is the negative of the rate constant for the conversion ofmaleamic acid to amine and anhydride, where A_(t) is the concentrationof maleamic acid at a time t and A₀ is the initial concentration ofmaleamic acid. For two separate experiments we calculated rate constantsof 0.066 sec⁻¹ and 0.157 sec⁻¹ which correspond to half lives of roughly10 and 4 minutes respectively.

B) Kinetics of conversion of dimethylmaleamic modified KL₃ (DM-KL₃) toKL₃. Dimethyl maleamic modified KL₃ (0.1 mg/mL) was incubated in 40 mMsodium acetate buffer pH 5 and 1 mM cetyltrimetylammonium bromide. Atvarious times, aliquots (10 μg) were removed and added to 0.05 mL of 1 Mborax solution containing 4 mM trinitrobenzenesulfonate. A half an hourlater, the absorbance of the solution at 420 nm was measured. Todetermine the concentration of amines at each time point, the extinctioncoefficient was determine for the product of trinitrobenzenesulfonateand poly-L-lysine. Using this extinction coefficient we were able tocalculate the amount of amines and maleamic groups at each time point. Aplot of ln (A_(t)/A₀) as a function of time was a straight line whoseslope is the negative of the rate constant for the conversion ofmaleamic acid to amine and anhydride, where A_(t) is the concentrationof maleamic acid at a time t and A₀ is the intial concentration ofmaleamic acid. We calculated a rate constant of 0.087 sec⁻¹ thatcorresponds to a half-life of roughly 8 minutes.

C) Kinetics of hydrolysis of glycolic acid ethoxylate(4 units)4-tert-buty-1,4-cyclohexadiene. Glycolic acid ethoxylate(4 units)4-tert-buty-1,4-cyclohexadiene (1 mg) was dissolved in placed into 1 mLof 15 mM sodium acetate pH 5 buffer. The absorbance of the solution at225 nm, which is the wavelength at which enol ethers absorb (Kresge, A.J.; Sagatys, D. S.; Chen, H. L. J. Am. Chem. Soc. 1977, 99, 7228) wasmeasured over time. A fit of the decrease of absorbance as a function oftime by an exponential decay function had a rate constant of 0.0159min⁻¹, which corresponds to a half-life of 40 minutes.

D) Kinetics of hydrolysis ofpoly(oxy-1-para-aceticphenoxymethylethylene-co-oxy-1-methylethylene).Poly(oxy-1-para-aceticphenoxymethylethylene-co-oxy-1-methylethylene)(0.16 mg/mL) was placed into 1 mL of 5 mM sodium acetate buffer pH 5.The absorbance of the solution at 225 nm was measured as a function oftime. The amount of time it took for the absorbance to decrease half ofmaximum was 37 minutes, i.e. the half-life of hydrolysis is 37 minutes.

E) Particle Formation ofpoly(oxy-1-para-aceticphenoxymethylethylene-co-oxy-1-methylethylene) asa Function of Acidification and Time. To a solution (0.5 mL) of 5 mMHEPES pH 8 was addedpoly(oxy-1-para-aceticphenoxymethylethylene-co-oxy-1-methylethylene) (54μg/mL) which had been incubated for various times in the presence of 1mM acetic acid (pH4–5), followed by the addition of polyallylamine. Theintensity of the scattered light and the size of the particle weremeasured (using a Brookhaven ZetaPlus Particle Sizer) as a function ofthe amount of time the polymer was incubated under acidic conditions.

Scattered light intensity Time at pH 4–5 (minutes) Size (nm) (kilocountsper second) 0 231 390 1 195 474 2 208 460 5 224 450 15  124  92 39  132250

F) Kinetics of Cleavage of Ketal. Synthesis of Microspheres ContainingAcid Labile Ketal Moieties:

F1) Esterification of Carboxylic Acid Modified Microspheres withDi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. To a suspensionof carboxylic acid modified microspheres (1000 μL, 2% solids, MolecularProbes) in H₂O (500 μL) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.0 mg, 36μmol, Aldrich Chemical Company), followed bydi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (23 mg, 73μmol), and the suspension was stirred at room temperature. After 16 hrs,the microspheres were removed by centrifugation. The supernatant wasremoved and the pellet was resuspended in 1.5 mL H₂O to wash. Themicrospheres were washed an additional 2×1.5 mL H₂O and suspended in 1mL H₂O.

F2) Aldehyde Derivatization of Esterified Carboxylic Acid ModifiedMicrospheres withDi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. To a solutionof succinic semialdehyde (3.7 mg, 36 μmol, Aldrich Chemical Company) inH₂O was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (8.7 mg, 46 μmol, Aldrich Chemical Company) followed byN-hydroxysuccinimide (5.3 mg, 46 μmol, Aldrich Chemical Company). Thesolution was stirred for 20 min at which time carboxylic acid modifiedmicrospheres esterified withdi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene (500 μL) wereadded. After 16 hrs, the microspheres were removed by centrifugation.The supernatant was removed and the pellet was resuspended in 1 mL H₂Oto wash. The microspheres were washed an additional 2×1 mL H₂O andsuspended in 1 mL H₂O. The aldehyde content of the microspheres wasdetermined on a 50 μL sample of the suspension with2,4-dinitrophenylhydrazine and NaBH₃CN. The absorbance measured at 349nm and fitted against a standard curve indicated 18 μmol of aldehydepresent in the reaction sample.

Attachment of Membrane Active Peptide to Acid Labile Moieties andLability Studies of these Systems:

F3) Attachment of a Peptide (Melittin) to the Aldehyde Derived fromCarboxylic Acid Modified Microspheres Esterified withDi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. To 100 μL ofthe aldehyde derivatized microshpere suspension was added 400 μL H₂O andmelittin (1 mg, 0.4 μmol, Mirus Corporation). After 12 hrs, NaBH₃CN (0.6mg, 9 μmol, Aldrich Chemical Company) was added. After 1 hr, thesuspension was centrifugated to pellatize the microspheres. Thesupernatant was removed and the pellet was resuspended in 1 mL H₂O towash. The microspheres were washed an additional 3×1 mL H₂O andsuspended in 1 mL H₂O. The last wash indicated the presence of activepeptide based on red blood cell lysis activity. The sample was washed1×25 mM HEPES, and 1×H₂O. The final wash was free of peptide based onred blood cell lysis assay.

F4) Blood Lysis Experiment on Melittin Conjugated to Microspheres viathe Di-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. Themicrospheres were taken up in H₂O (500 μL) and partitioned into five 100μL samples. Four of the samples were diluted to 1000 μL with sodiumphosphate buffer (100 mM) at pH 7.5, 6.0, 5.5, and 5.0. Samples wereheld at 37° C., spun down, and 150 μL aliquots taken at 30 min, 60 min,90 min, and 16 hrs. A portion of each sample (100 μL) was diluted withsodium phosphate buffer (400 μL, pH 7.5) and added to red blood cells(100 μL, pH 7.5). Red blood cell lysis was measured after 10 min bymeasuring the absorbance at 541 nm.

A control sample was also measured in which 100% of the red blood cellshad been lysed with melittin alone.

Sample A₅₄₁ Blood 0.026 100% lysis 1.881 30 min pH 7.5 0.026 30 min pH6.0 0.326 30 min pH 5.5 0.609 30 min pH 5.0 0.659 60 min pH 7.5 0.027 60min pH 6.0 0.212 60 min pH 5.5 0.526 60 min pH 5.0 0.730 90 min pH 7.50.036 90 min pH 6.0 0.390 90 min pH 5.5 0.640 90 min pH 5.0 0.892 16 hrspH 7.5 0.065 16 hrs pH 6.0 0.354 16 hrs pH 5.5 0.796 16 hrs pH 5.0 1.163The fifth 100 μL sample was further divided into 25 μL samples, three ofwhich were diluted to 250 μL with sodium phosphate buffer (100 mM) at pH7.5, 6.0, and 5.0. The samples were held at 37° C. for 30 min, spun downand the supernatant removed, and resuspended in 2.5 M NaCl solution (50μL) and mixed. After 10 min the microspheres were spun down and thesupernatant removed. The samples were added to red blood cells (500 μL,100 mM) and the absorbance was measured at 541 nm.

Sample A₅₄₁ Blood (NaCl wash) 0.041 pH 7.5 0.053 pH 7.5 (NaCl wash)0.087 pH 6.0 0.213 pH 6.0 (NaCl wash) 0.162 pH 5.0 0.685 pH 5.0 (NaClwash) 0.101The results indicate that under acidic conditions, the modified peptideis released from the microsphere and is available to interact with thecell membrane as indicated by the red blood cell lysis. The resultsindicate that the modified peptide is not released at pH 7.5.Additionally, the lysis activity results indicate the release ofmodified peptide is rapid at all acidic pH levels tested (t<30 min) withslow continual release thereafter, and that more modified peptide isreleased at lower pH (larger red blood cell lysis). The results alsoindicate that more modified peptide is released upon washing themicrosphere with a salt solution.

F5) Attachment of a Peptide (Melittin) to the Aldehyde Derived fromPoly-Glutamic Acid Partially Esterified withDi-(2-methyl-4-hydroxymethyl-1,3-dioxolane)-1,4-benzene. To a solutionof the aldehyde-poly-glutamic acid compound (1.0 mg, 7.7 μmol) in water(200 μL) was added melittin (4.0 mg, 1.4 μmol) and the reaction mixturewas stirred at room temperature. After 12 hrs the reaction mixture wasdivided into two equal portions. One sample (100 μL) was dialyzedagainst 1% ethanol in water (2×1 L, 12,000–14,000 MWCO) and testedutilizing a theoretical yield of 1.7 mg. To the second portion (100 μL)was added sodium cyanoborohydride (1.0 mg, 16 μmol, Aldrich ChemicalCompany). The solution was stirred at room temperature for 1 hr and thendialyzed against water (2×1 L, 12,000–14,000 MWCO). The resultingmaterial was utilized assuming a theoretical yield of 1.7 mg ofconjugate.

Lability of Polymers Containing Acid Labile Moieties:

F6) Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal Acid ofPolyvinylphenyl Ketone and Glycerol Ketal Complexes. Particle sizing(Brookhaven Instruments Corporation, ZetaPlus Particle Sizer, I90, 532nm) indicated an effective diameter of 172 nm (40 μg) for the ketalacid. Addition of acetic acid to a pH of 5 followed by particle sizingindicated a increase in particle size to 84000 nm. A poly-L-lysine/ketalacid (40 μg, 1:3 charge ratio) sample indicated a particle size of 142nm. Addition of acetic acid (5 μL, 6 N) followed by mixing and particlesizing indicated an effective diameter of 1970 nm. This solution washeated at 40° C. Particle sizing (by a Brookhaven ZetaPlus ParticleSizer) indicated an effective diameter of 74000 nm and a decrease inparticle counts.

Results: The particle sizer data indicates the loss of particles uponthe addition of acetic acid to the mixture.

F7) Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal fromPolyvinyl Alcohol and 4-Acetylbutyric Acid Complexes. Particle sizing(Brookhaven Instruments Coporation, ZetaPlus Particle Sizer, I90, 532nm) indicated an effective diameter of 280 nm (743 kcps) forpoly-L-lysine/ketal from polyvinyl alcohol and 4-acetylbutyric acidcomplexes (1:3 charge ratio). A poly-L-lysine sample indicated noparticle formation. Similarly, a ketal from polyvinyl alcohol and4-acetylbutyric acid sample indicated no particle formation. Acetic acidwas added to the poly-L-lysine/ketal from polyvinyl alcohol and4-acetylbutyric acid complex to a pH of 4.5. Particle sizing (by aBrookhaven ZetaPlus Particle Sizer) indicated particles of 100 nm, butat a low count rate (9.2 kcps). Results: The particle size dataindicates the loss of particles upon the addition of acetic acid to themixture.

F8) Size Exclusion Chromatography and Acid Lability of MC228. MC208 (1.5mg) was taken up in 50 mM HEPES (0.3 mL, pH 8.5) and passed through aSephadex G50 column (8 cm column, 50 mM HEPES (pH 8.5) eluent) and 0.5mL fractions were collected. The absorbance of the fractions wasdetermined at 300 nm. Two additional samples (1.5 mg) were prepared in50 mM Citrate buffer at pH 2 and pH 5 (0.3 mL) and allowed to sit a roomtemperature for 45 min prior to running on the Sephadex G50 column (8 cmcolumn, 50 mM HEPES (pH 8.5) eluent). The absorbance of the fractionswas determined at 300 nm.

Fraction number pH 8.5 pH 5 pH 2 1 0.018 0.040 0.022 2 0.024 0.019 0.0133 0.019 0.015 0.008 4 0.028 0.118 0.024 5 0.287 0.527 0.293 6 1.0910.693 0.604 7 0.976 0.818 0.715 8 0.888 1.071 0.895 9 0.907 1.178 1.08210 0.944 1.289 1.298 11 0.972 1.296 1.423 12 0.941 1.212 1.326 13 0.9130.924 1.140 14 0.764 0.640 1.012 15 0.589 0.457 0.841 16 0.415 0.2640.655Results: The column demonstrates that upon incubating the sample underacidic conditions, the molecular weight of the polymer is decreasedindicating the polymer is labile under acidic conditions.

F9) Acid Lability of MC208. A sample of MC208 in dimethylformamide (20μL) was divided into four equal samples. To each sample was addedcitrate buffer (100 μL, pH 4) and the resulting samples (final pH of 5)were incubated at 37° C. for 2, 4, 8, and 24 hrs. The samples were thenanalyzed by thin layer chromatography against a sample not exposed toacidic conditions.

The results indicated increasing amounts of higher Rf material withincreasing time, indicated degredation of the polymer.

F10) Particle Sizing and Acid Lability of pDNA (pCI Luc)/MC208Complexes. Particle sizing (Brookhaven Instruments Coporation, ZetaPlusParticle Sizer, I90, 532 nm) indicated an effective diameter of 293 nm(687 kcps) for pDNA (25 μg pDNA)/di-(2-methyl-4-hydroxymethyl(succinicsemialdehyde ester)-1,3-dioxolane)-1,4-benzene:1,4-bis(3-aminopropyl)-piperazine copolymer complexes (1:3 chargeratio). HCl was added to the complex to approximately pH 5 and theparticle size was measured. The reading indicated particles with aneffective diameter of 11349 nm (120 kcps).

Results: The particle size data indicates MC208 compacts pDNA into smallparticles. The results also indicate the loss of particles upon theaddition of HCl to the mixture by floculation.

G) Kinetics of Cleavage of Imine: Particle Sizing and Acid Lability ofpDNA (pCl Luc)/1,4-Bis(3-aminopropyl)piperazine Glutaric DialdehydeCopolymer Complexes. To 50 μg pDNA in 2 mL HEPES (25 mM, pH 7.8) wasadded 135 μg 1,4-bis(3-aminopropyl)piperazine glutaric dialdehydecopolymer. Particle sizing (Brookhaven Instruments Coporation, ZetaPlusParticle Sizer, I90, 532 nm) indicated an effective diameter of 110 nmfor the complex. A 50 μg pDNA in 2 mL HEPES (25 mM, pH 7.8) sampleindicated no particle formation. Similarly, a 135 μg1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer in 2 mLHEPES (25 mM, pH 7.8) sample indicated no particle formation. Aceticacid was added to the pDNA (pCI Luc)/1,4-bis(3-aminopropyl)piperazineglutaric dialdehyde copolymer complex to a pH of 4.5. Particle sizingindicated particles of 2888 nm, and aggregation was observed.

Results: 1,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde copolymercondenses pDNA, forming small particles. Upon acidification, theparticle size increases, and aggregation occurs, indicating cleavage ofthe polymeric imine.

Example 5

Hemolysis Assay

A) Lysis of Erythrocytes by the peptides Melittin and KL₃ and theirdimethylmaleamic acid derivatives as a function of pH. Themembrane-disruptive activity of the peptide melittin and subsequentblocking of activity by anionic polymers was measured using a red bloodcell (RBC) hemolysis assay. RBCs were harvested by centrifuging wholeblood for 4 min. They were washed three times with 100 mM dibasic sodiumphosphate at the desired pH, and resuspended in the same buffer to yieldthe initial volume. They were diluted 10 times in the same buffer, and200 uL of this suspension was used for each tube. This yields 10^8 RBCsper tube. Each tube contained 800 uL of buffer, 200 uL of the RBCsuspension, and the peptide with or without polymer. Each sample wasthen repeated to verify reproducibility. The tubes were incubated for 30minutes in a 37° C. water bath. They were spun for 5 min at full speedin the microcentifuge. Lysis was determined by measuring the absorbanceof the supernatant at 541 nm, reflecting the amount of hemoglobin thathad been released into the supernatant. Percent hemolysis was calculatedassuming 100% lysis to be measured by the hemoglobin released by the redblood cells in water; controls of RBCs in buffer with no peptide werealso run.

Percent Hemolysis Peptide pH 5.4 pH 7.5 Unmodified Peptides KL₃ 86, 77,86 54, 77, 54 Melittin 85 92 Dimethylmaleamic Derivatives KL₃ 30, 55, 268, 3, 2 Melittin 100 1 Succinyl Derivatives KL₃ 2, 2, 2 1, 1, 2 Melittin5 2

B) Lysis of Erythrocytes by Poly Propacrylic Acid and subsequentblocking of activity by cationic polymers with reversible blocking ofactivity with cleavable disulfide cations in the presence ofGlutathione. The pH-dependent membrane-disruptive activity of the PPAAcand subsequent blocking of activity by cationic polymers was measuredusing a red blood cell (RBC) hemolysis assay. RBCs were harvested bycentrifuging whole blood for 4 min. They were washed three times with100 mM dibasic sodium phosphate at the desired pH, and resuspended inthe same buffer to yield the initial volume. They were diluted 10 timesin the same buffer, and 200 L of this suspension was used for each tube.This yields 10^8 RBCs per tube. Each tube contained 800 L of buffer, 200L of the RBC suspension, and the polymer. Each sample was done intriplicate, and was then repeated to verify reproducibility. The tubeswere incubated for an hour and a half in a 37° C. water bath. They werespun for 5 min at full speed in the microcentifuge. Lysis was determinedby measuring the absorbance of the supernatant at 541 nm, reflecting theamount of hemoglobin which had been released into the supernatant.Percent hemolysis was calculated assuming 100% lysis to be measured bythe hemoglobin released by the red blood cells in water; controls ofRBCs in buffer with no polymer were also run.

Results at pH 6.0: Mock: 3% PPAAc: 98% PPAAc + p-L-Lysine 3% PPAAc +p-L-Lysine w/1 mM Glutathione 2% PPAAc + 5,5′-Dithiobis(2-nitrobenzoicacid)- 12% 1,4-Bis(3-aminopropyl)piperazine Copolymer PPAAc +5,5′-Dithiobis(2-nitrobenzoic acid)- 98%1,4-Bis(3-aminopropyl)piperazine Copolymer w/1 mM Glutathione PPAAc +L-cystine − 1,4-bis(3-aminopropyl)piperazine copolymer 2% PPAAc +L-cystine − 1,4-bis(3-aminopropyl)piperazine copolymer 20% w/1 mMGlutathione

C) Lysis of Erythrocytes by the peptide Melittin or KL3 and subsequentblocking of activity by anionic polymers or modification withdimethylmaleic anhydride. The membrane-disruptive activity of thepeptide melittin and subsequent blocking of activity by anionic polymerswas measured using a red blood cell (RBC) hemolysis assay. RBCs wereharvested by centrifuging whole blood for 4 min. They were washed threetimes with 100 mM dibasic sodium phosphate at the desired pH, andresuspended in the same buffer to yield the initial volume. They werediluted 10 times in the same buffer, and 200 uL of this suspension wasused for each tube. This yields 10^8 RBCs per tube. Each tube contained800 uL of buffer, 200 uL of the RBC suspension, and the peptide with orwithout polymer. Each sample was then repeated to verifyreproducibility. The tubes were incubated for 30 minutes in a 37° C.water bath. They were spun for 5 min at full speed in themicrocentifuge. Lysis was determined by measuring the absorbance of thesupernatant at 541 nm, reflecting the amount of hemoglobin that had beenreleased into the supernatant. Percent hemolysis was calculated assuming100% lysis to be measured by the hemoglobin released by the red bloodcells in water; controls of RBCs in buffer with no peptide were alsorun.

Results at pH 7.5: Mock: 1% Melittin 100% Melittin + pAcrylic Acid 9%DM-Melittin 1% DM-Melittin post incubation at pH 4 30 seconds 100% KL386% DM-KL3 4% DM-KL3 post incubation at pH 5.4 30 seconds 85%

Example 6

Endosome Lysis

Endosome Disruption Assay: with Dimethylmaleamic-modified melittin. HeLacells were plated in 6-well tissue culture dishes containing microscopeslide coverslips and grown in Delbecco's Modified Eagle's Medium(DMEM)+10% fetal calf serum+penn/strep for 24–48 hours until 30–60%confluent. Growth media was aspirated and 1 ml pre-heated (37° C.)serum-free DMEM+2 mg/ml fluorescein isothiocyanate (FITC) labeleddextran(10 kDa)±50 μg DM-melittin or 50 μg melittin was added to thecells and incubated at 37° C. in a humidified CO₂ incubator. After 25min, media containing FITC-dextran±melittin was removed, the cells werewashed twice with 1 ml 37° C. DMEM lacking FITC-dextran and melittin,and cells were incubated for an additional 35 min at 37° C. in 1 mlfresh DMEM. In order to assess possible cell lysis caused by melittin,propidium iodide was added for the final 5 min of incubation. Propidiumiodide is impermeable to the cell plasma membrane and thus does notstain live cells. However, if the plasma membrane has been damaged,propidium iodide enters the cell where it will brightly stain thenucleus. To process slides for analysis, cells were washed 3 times withcold phosphate buffered saline (PBS), fixed in PBS+4% formaldehyde for20–30 min at 4° C., and washed again 3 times with cold PBS. Excessliquid was drained from coverslips which were then mounted onto glassslides. Fluorescence was then analyzed on a Zeiss LSM510 confocalmicroscope. FITC was excited by a 488 nm argon laser and fluorescenceemission was detected by a long pass 505 nm filter. FITC-dextran thathad been internalized but not released from internal vesicles/endosomesappeared as a punctate cytoplasmic signal. In the presence ofDM-melittin, a loss of punctate cytoplasmic signal was observed with aconcomitant appearance of a diffuse cytoplasmic signal, indicative ofrelease of dextran from endosomes. For cells incubated with unmodifiedmelittin near 100% cell death was observed as determined by propidiumiodide staining of nuclei and loss of cells from the sample.

Example 7

In Vivo Circulation Studies. General procedure for the reaction ofpoly-L-Lysine compacted DNA particles polyethylene glycol methyl ether2-propionic-3-methylmaleate (CDM-PEG). Plasmid DNA (200 μg/ml) in 290 mMGlucose/5 mM Hepes pH8 was compacted with poly-L-Lysine (mw: 52,000)(144 μg/ml). This particle is then reacted with 0.5, 1, 2 or 5-foldweight excess of CDM-PEG to amines on the poly-L-lysine.Effect of CDM-PEG modified poly-L-lysine:DNA particles in vivo. PlasmidDNA labeled with Cy3 Label IT(Mirus Corporation, Madison, Wis.) wascompacted into a particle with a 1.2 fold charge excess of poly-L-lysine(mw: 52,000). The particles were then reacted with either a non-reactivePolyethylene Glycol (mw: 5000) or with amine-reactive CDM-PEG at a 0.5molar equivalent to amines on the poly-L-lysine. 50 μg aliquots of DNAwere injected into the tail vein of male ICR mice of approximately 20grams in weight. Blood was taken at one hour and the smears wereinspected for Cy3 fluorescence still in circulation. The animals werethen sacrificed and the liver, lung, kidney and spleen were harvestedand snap frozen for cryosectioning and the resulting slices wereinspected for Cy3 fluorescence.Results:The animal injected with the fluorescent particles treated withnon-reactive Polyethylene Glycol showed no fluorescence in circulationin the blood at one hour and very little fluorescence in the liver,kidney or spleen, leaving the significant portion of fluorescence in thelung. The animal injected with the fluorescent particles treated withCDM-PEG showed a high level of fluorescence still in circulation in theblood at one hour and also had a high level of fluorescence evenlyspread throughout the liver, some spread in the kidney and spleen, withlittle fluorescence in the lung.Various Compounds which may be Utilized in the System Provided:

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in cell biology, chemistry,molecular biology, biochemistry or related fields are intended to bewithin the scope of the following claims.

1. A process for transfecting a nucleic acid into a cell in vivo,comprising: a) attaching a membrane activity inhibitor to a membraneactive peptide via a labile linkage, wherein the inhibitor is detachedwithin the cell; b) adding the peptide to a solution containing thenucleic acid; c) delivering the peptide and nucleic acid to the cell,wherein the peptide and the nucleic acid are endocytosed; and, d)transfecting the cell.
 2. The process of claim 1 wherein the peptideconsists of pardaxin.
 3. The process of claim 1 wherein the peptideconsists of KL3.
 4. The process of claim 1 wherein the peptide consistsof magainin.
 5. The process of claim 1 wherein the labile linkage isselected from the group consisting of pH-labile, very pH labile, andextremely pH-labile.
 6. The process of claim 1 wherein the labilelinkage is selected from the group consisting of disulfide, acetal,ketal, enol ether, enol esters amide, imine, imminium, enamine, allylether, silazane, and silyl enol ether bonds.
 7. The process of claim 1wherein the labile linkage is selected from the group consisting ofdials, diazo, ester, sulfone, and silicon-carbon bonds.
 8. A process fortransfecting a nucleic acid into a cell in vivo, comprising: a)attaching a reversible labile membrane activity inhibitor to a melittinpeptide wherein the inhibitor is detached upon association with thecell; b) adding the peptide to a solution containing the nucleic acid;c) contacting the peptide and nucleic acid with the cell, wherein thepeptide and nucleic acid are endocytosed; and, d) transfecting to cell.9. A process for transfecting a nucleic acid into a cell in vivo,comprising: a) attaching a membrane activity inhibitor to a membraneactive polymer via a labile linkage wherein the inhibitor is detachedupon association with the cell; b) adding the membrane active polymer toa solution containing the nucleic acid; c) contacting the membraneactive polymer and nucleic acid with the cell wherein the membraneactive polymer and the nucleic acid are endocytosed; and, d)transfecting the cell.
 10. The process of claim 9 wherein the labilelinkage is selected from the group consisting of pH-labile, very pHlabile, and extremely pH-labile.
 11. The process of claim 9 wherein thelabile linkage is selected from the group consisting of disulfide,acetal, ketal, enol ether, enol ester, amide, imine, imminium, enamine,silyl ether, silazane, and silyl enol ether bonds.
 12. The process ofclaim 9 wherein to labile linkage is selected from the group consistingof diols, diazo, ester, sulfone, and silicon-carbon bonds.